In situ hybridization

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Boom, R.; Geelen, J. L.; Sol, C. J.; Raap, A. K.; ... Raap, A. K.; Van de Rijke, F .M.; Dirks, R. W.; Sol, C. J.; ...... Schöfer, C.; Müller, M.; Leitner, M. D.; Wachtler, F.
General Introduction to In Situ H ybridization

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General Introduction to In Situ Hybridization I n situ hybridization techniques allow specific nucleic acid sequences to be detected in morphologically preserved chromosomes, cells or tissue sections. In combination with immunocytochemistry, in situ hybridization can relate microscopic topological information to gene activity at the D N A, mRN A, and protein level. The technique was originally developed by Pardue and G all (1969) and (independently) by John et al. (1969). At this time radioisotopes were the only labels available for nucleic acids, and autoradiography was the only means of detecting hybridized sequences. Furthermore, as molecular cloning was not possible in those days, in situ hybridization was restricted to those sequences that could be purified and isolated by conventional biochemical methods (e.g., mouse satellite DN A, viral DN A, ribosomal RN As).

In spite of the high sensitivity and wide applicability of in situ hybridization techniques, their use has been limited to research laboratories. This is probably due to the problems associated with radioactive probes, such as the safety measures required, limited shelf life, and extensive time required for autoradiography. In addition, the scatter inherent in radioactive decay limits the spatial resolution of the technique. H owever, preparing nucleic acid probes with a stable nonradioactive label removes the major obstacles which hinder the general application of in situ hybridization. Furthermore, it opens new opportunities for combining different labels in one experiment. The many sensitive antibody detection systems available for such probes further enhances the flexibility of this method. In this manual, therefore, we describe nonradioactive alternatives for in situ hybridization.

Molecular cloning of nucleic acids and improved radiolabeling techniques have changed this picture dramatically. For example, D N A sequences a few hundred base pairs long can be detected in metaphase chromosomes by autoradiography (H arper et al., 1981; Jhanwag et al., 1984; Rabin et al., 1984; Schroeder et al., 1984). Also radioactive in situ techniques can detect low copy number mRN A molecules in individual cells (H arper et al., 1986). Some years ago, chemically synthesized, radioactively labeled oligonucleotides began to be used, especially for in situ mRN A detection (Coghlan et al., 1985).

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G eneral I ntroduction to I n Situ H ybridiz ation

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Direct and indirect methods There are two types of nonradioactive hybridization methods: direct and indirect. In the direct method, the detectable molecule (reporter) is bound directly to the nucleic acid probe so that probe-target hybrids can be visualized under a microscope immediately after the hybridization reaction. For such methods it is essential that the probe-reporter bond survives the rather harsh hybridization and washing conditions. Perhaps more important, however, is, that the reporter molecule does not interfere with the hybridization reaction. The terminal fluorochrome labeling procedure of RN A probes developed by Bauman et al. (1980, 1984), and the direct enzyme labeling procedure of nucleic acids described by Renz and Kurz (1984) meet these criteria. Boehringer Mannheim has introduced several fluorochrome-labeled nucleotides that can be used for labeling and direct detection of DN A or RN A probes. If antibodies against the reporter molecules are available, direct methods may also be converted to indirect immunochemical amplification methods (Bauman et al., 1981; Lansdorp et al., 1984; Pinkel et al., 1986). Indirect procedures require the probe to contain a reporter molecule, introduced chemically or enzymatically, that can be detected by affinity cytochemistry. Again, the presence of the label should not interfere with the hybridization reaction or the stability of the resulting hybrid. The reporter molecule should, however, be accessible to antibodies. A number of such hapten modifications has been described (Langer et al., 1981; Leary et al., 1983; Landegent et al., 1984; Tchen et al., 1984; H opman et al., 1986; H opman et al., 1987; Shroyer and N akane, 1983; Van Prooijen et al., 1982; Viscidi et al., 1986; Rudkin and Stollar, 1977; Raap et al., 1989). O ne of the most popular is the biotin-streptavidin system. Boehringer Mannheim offers an alternative, the Digoxigenin (DIG/Genius™) System which is described in detail later in this chapter.

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A few years ago, the chemical synthesis of oligonucleotides containing functional groups (e.g., primary aliphatic amines or sulfhydryl groups) was described. These can react with haptens, fluorochromes or enzymes to produce a stable probe which can be used for in situ hybridization experiments (Agrawal et al., 1986; C hollet and Kawashima, 1985; H aralambidis et al., 1987; Jablonski et al., 1986). Modified oligonucleotides can also be obtained with the D IG / G enius™ system (Mühlegger et al., 1990). Such oligonucleotide probes will undoubtedly be widely used as automated oligonucleotide synthesis makes them available to researchers not familiar with D N A recombinant technology. This manual concentrates on two labeling systems: ! Indirect methods using digoxigenin (detected by specific antibodies) and biotin (detected by streptavidin) ! D irect methods using fluorescein or other fluorochromes directly coupled to the nucleotide The ordering information in C hapter 7 lists all the kits and single reagents Boehringer Mannheim offers for nonradioactive labeling and detection.

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Guidelines for In Situ H ybridization

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Guidelines for In Situ Hybridization An in situ hybridization protocol follows this general outline: ! Preparation of slides and fixation of material ! Pretreatments of material on slides, e.g., permeabilization of cells and tissues ! D enaturation of in situ target D N A (not necessary for mRN A target) ! Preparation of probe ! I n situ hybridization ! Posthybridization washes ! Immunocytochemistry ! Microscopy

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All these steps are discussed below in detail. The information provided will make it easier to decide whether a certain step should or should not be included in a given in situ hybridization protocol.

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Details of the Technique Slide preparation

Pretreatments of material on the slide

For chromosome spreads, alcohol/ ether (1:1) cleaned slides are sufficient. H owever, since tissue sections may be lost during the procedure, use either polylysine or glutaraldehyde-activated gelatin chrome aluminum slides for these sections.

Endogenous enzyme inactivation

Fixation To preserve morphology, the biological material must be fixed. From a chemical point of view, there is little limitation in the type of fixation used because one of the following will be true: ! The functional groups involved in base pairing are protected in the double helix structure of duplex D N A. ! RN A is fairly unreactive to crosslinking agents. ! The reaction is reversible (e.g., with formaldehyde). For metaphase chromosome spreads, methanol/acetic acid fixation is usually sufficient. For paraffin-embedded tissue sections, use formalin fixation. Cryostat sections fixed for 30 min with 4% formaldehyde or with Bouin’s fixative have been used successfully, as well as paraformaldehyde vapor fixation. Tissues can also be freeze-dried. It should be noted that the D N A and RN A target sequences are surrounded by proteins and that extensive crosslinking of these proteins masks the target nucleic acid. Therefore, permeabilization procedures are often required. U nfortunately, a fixation protocol which can be used for all substrates has not yet been described. The fixation and pretreatment protocols must be optimized for different applications.

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When an enzyme is used as the label, the endogenous enzyme activity may have to be inactivated. For peroxidase, this is done by treating the sections with 1% H 2O 2 in methanol for 30 min. For alkaline phosphatase, levamisole may be added to the substrate solution, but this may be unnecessary since residual alkaline phosphatase activity is usually lost during hybridization.

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RNase treatment

RN ase treatment serves to remove endogenous RN A and may improve the signal-tonoise ratio in DN A-DN A hybridizations. This treatment can also be used as a control in hybridizations with (m)RN A as target. It is done by incubating the preparations in DN ase-free RN ase (100 µg/ml) in 2 x SSC at 37°C for 60 min. (SSC = 150 mM N aCl, 15 mM sodium citrate, pH 7.4). HCl treatment

In several protocols a 20–30 min treatment with 200 mM H C l is included. The precise action of the acid is not known, but extraction of proteins and partial hydrolysis of the target sequences may contribute to an improvement in the signal-to-noise ratio. Detergent treatment

Preparations may be pretreated with Triton ® X-100, sodium dodecyl sulfate, or other detergents if lipid membrane components have not been extracted by other procedures such as fixation, dehydration, embedding, and endogenous enzyme inactivation procedures.

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Protease treatment

Protease treatment serves to increase target accessibility by digesting the protein that surrounds the target nucleic acid. To digest the sample, incubate the preparations with up to 500 µg/ ml Proteinase K (the optimal amount must be determined) in 20 mM Tris-H C l, 2 mM C aC l2, pH 7.4, for 7.5–30 min at 37°C . For example, with chromosomes or isolated preparations of nuclei, a reasonable starting point for protease digestion is up to 1 µg/ ml Proteinase K for 7.5 min. For formalin-fixed material, 5–15 µg/ ml for 15–30 min usually gives good results.

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The use of pepsin has been shown to give excellent results for formalin-fixed, paraffinembedded tissue sections. Routine pepsin digestion involves incubating the preparations for 30 min at 37°C in 200 mM H C l containing 500 µg/ ml pepsin. For the pretreatment of chromosome spreads with pepsin see page 70. O ther hydrolases, such as C ollagenase and diastase, may be tried if preparations of connective tissue and liver give high background reactions. Also, freeze/ thaw cycles have been used to improve the penetration of probes into tissues.

Prehybridization A prehybridization incubation is often necessary to prevent background staining. The prehybridization mixture contains all components of a hybridization mixture except for probe and dextran sulfate.

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Denaturation of probe and target For in situ hybridization to chromosomal D N A, the D N A target must be denatured. In general such treatments may lead to loss of morphology, so in practice, a compromise must be found between hybridization signal and morphology. Alkaline denaturations have traditionally been used. H eat denaturations have also become popular, because of their experimental simplicity and greater effectiveness. Variations in time and temperature should be evaluated to find the best conditions for denaturation. For heat denaturation, the probe and target chromosomal D N A may be denatured simultaneously. To accomplish this, put the probe on the slide and cover with a coverslip. Bring the slide to 80°C for 2 min in an oven for the denaturation and then cool to 37°C . For tissue sections, if necessary, extend the time of denaturation at 80°C to 10 min. For competition in situ hybridization, where the labeled probe is allowed to reanneal with unlabeled competitor D N A, denature the chromosomal preparation and probe separately.

Hybridization See C hapter 3 for the composition of the hybridization solution, and the factors affecting hybridization kinetics and hybrid stability.

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Fluorescence

Posthybridization washes Labeled probe can hybridize nonspecifically to sequences which are partially but not entirely homologous to the probe sequence. Such hybrids are less stable than perfectly matched hybrids. They can be dissociated by performing washes of various stringencies (as described in Chapter 3). The stringency of the washes can be manipulated by varying the formamide concentration, salt concentration, and temperature. O ften a wash in 2 x SSC containing 50% formamide suffices. For some applications the stringency of the washes should be higher. H owever, we recommend hybridizing stringently rather than washing stringently.

Immunocytochemistry In this manual immunocytochemical procedures using digoxigenin, biotin, and fluorochromes are described. These allow flexibility in the choice of the final reporter molecule and type of microscopy. Blocking reaction

U se a blocking step prior to the immunological procedure to remove high background. For example, perform biotin detection steps in PBS containing Tween ® 20 and BSA (when using monoclonal antisera) or normal serum (when using polyclonal antisera). Perform routine digoxigenin detection in Tris-H C l buffer containing Blocking Reagent (instead of BSA or normal serum) to remove background. Also the addition of 0.4 M N aC l can help prevent background staining if the antigen/ hapten bond can survive the high salt conditions. In a standard reaction, block the target tissue, cell, or chromosome preparation and then incubate it with the antibodyconjugate solution for 30 min at 37°C (or 2 h at room temperature) in a moist chamber. Afterwards, wash the slide three times (5–10 min each) with buffer containing Tween ® 20.

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Useful fluorochromes for fluorescence in situ hybridization (FISH ) include the blue fluorochrome AMCA (amino-methylcoumarin-acetic acid), the green fluorophore fluorescein, and the red fluorochromes CY3, rhodamine, and Texas Red™. Recently, the FISH application of an infrared dye has been reported (Ried et al., 1992), although it can only be visualized with infrared-sensitive cameras. For routine experiments, several immunofluorophores with good spectral separation properties can be used for FISH analysis (Table 1). Chapter 5 contains detailed procedures for using all these fluorochromes.

AMC A Fluorescein C Y3 Rhodamine Texas Red

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Color

Excitation max. (nm)

Emission max. (nm)

Blue G reen Red Red Red

399 494 552 555 590

446 523 565 580 615 "

Rhodamine-, fluorescein-, and coumarinbased dyes cover the three primary colors of the visible part of the electro-magnetic spectrum. By using selective excitation and emission filters, and dichroic mirrors, these colors can be visualized without “crosstalk”, making triple color FISH feasible. To increase the identification of targets by color, a combinatorial labeling approach has been developed (N iederlof et al., 1990; Ried et al., 1992; Wiegant et al., 1993). Multiplicity is then given by 2n – 1, where n is the number of colors.

Table 1: Spectral properties of fluorophores used in FISHanalysis.

After establishing that hybridized probes labeled with two haptens in different ratios still fluoresce at fairly constant ratios, laboratories extended the combinatorial approach to ratio-labeling (N ederlof et al., 1992). In ratio-labeling, the ratio of fluorescence intensities of the various color combinations is used for color identification of the probes. This approach allowed simultaneous identification of twelve different probes (D auwerse et al., 1992). For detailed information, refer to the literature describing combinatorial and ratio labeling (Wiegant and D auwerse, 1995).

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Fluorescent DN A counterstaining is usually performed with red fluorescent propidium iodide (PI) or with blue fluorescent DAPI. Currently, new counterstains are being introduced, e.g., green fluorescent YO YO -1 (Molecular Probes, Inc., USA). An antifading agent should be added to the embedding medium to retard fading. When fluorescent labels are used, the recommended antifading agent is Vectashield (Vector Laboratories). The different D N A counterstains can be directly dissolved in the embedding medium (40 ng D API/ ml; 100 ng PI/ ml; or 0.1 µM YO YO -1/ ml) or the fluorescent specimen can first be counterstained and then embedded in Vectashield. If desired, the coverslip may be sealed with nail varnish.

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Enzymes

U se any enzyme commonly used in immunocytochemistry. We show examples for peroxidase and alkaline phosphatase. With peroxidase (PO D), use the diaminobenzidine (DAB)/imidazole reaction (Graham and Karnovsky, 1966). With alkaline phosphatase (AP), use the 5-Bromo-4-chloro-3indolylphosphate/N itro-blue tetrazolium (BCIP/N BT) reaction. The advantages of these colorimetric detection methods are good localization properties, high sensitivity (De Jong et al., 1985; Straus, 1982; Scopsi and Larson, 1986) and stability of the precipitates. As the D AB reaction produces a color which contrasts with the blue alkaline phosphatase staining reaction (and vice versa), one can use both enzymes in double hybridizations. Furthermore, both detection methods have bright reflective properties. They can be amplified by gold/ silver (G allyas et al., 1982; Burns et al., 1985) and the color can be modified with heavy metal ions (H su and Soban, 1982; C remers et al., 1987).

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When using brightfield microscopy with peroxidase, prepare the following peroxidase substrate solution: 0.5 mg/ ml diaminobenzidine in 50 mM Tris-H C l, pH 7.4, containing 10 mM imidazole. Shortly before use, add 0.05% H 2O 2. Incubate with substrate from 2–30 min (depending on the signal-to-noise ratio). We advise inspecting sections microscopically during the peroxidase reaction. Stop the reaction by washing the sample with water. C ounterstain with G iemsa if desired. In the case of reflection contrast microscopy with peroxidase, prepare the following peroxidase substrate solution: 0.1 mg/ ml diaminobenzidine in 50 mM Tris-H C l, pH 7.4. Shortly before use, add 0.01% H 2O 2. The usual reaction time is 10 min. After stopping the reaction with H 2O and dehydrating with ethanol, evaluate slides by oil-immersion microscopy without embedding. When using Alkaline Phosphatase, prepare the following substrate: 0.16 mg/ml 5-Bromo4-chloro-3-indolyl-phosphate (BC IP) and 0.33 mg/ ml N itro-blue tetrazolium salt (N BT) in 200 mM Tris-H C l, 10 mM MgC l2, pH 9.2. D etermine reaction times by evaluating the signal-to-noise ratio, which should be checked microscopically during the enzyme reaction. The reaction medium itself is stable (in the dark). The final product (N BT formazan) also has reflective properties. A recently introduced alkaline phosphatase substrate (H N PP/ Fast Red TR) also allows fluorescent detection of alkaline phosphatase label. For fluorescence microscopy, prepare the following substrate: 10 mg/ ml H N PP (in dimethylformamide) and 25 mg/ ml Fast Red TR in redist H 2O . For details on the use of H N PP/ Fast Red TR, see the article in C hapter 5, page 108.

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Microscopy Brightfield microscopy

In brightfield microscopy the image is obtained by the direct transmission of light through the sample. Evaluation of in situ hybridization results by brightfield microscopy is preferred for most routine applications because the preparations are permanent. H owever, the sensitivity demanded by many applications (e.g., single copy gene localization requires the detection of a few attograms of D N A) may require more sophisticated microscopy. Darkfield microscopy

In the darkfield microscope, the illuminating rays of light are directed from the side, so that only scattered light enters the microscope lenses. C onsequently, the material appears as an illuminated object against a black background. D arkfield microscopy is extensively used for radioactive in situ hybridization experiments because large fields can be examined at low magnification. The distribution of the silver grains can be seen with high contrast. In contrast, these types of microscopy are seldom used to localize reaction products of nonradioactive hybridization procedures (H eyting et al., 1985). H owever, Garson et al. (1987) shows the application of phase contrast microscopy to single copy gene detection. Phase contrast microscopy

Phase contrast microscopy exploits the interference effects produced when two sets of waves combine. This is the case when light passing e.g., through a relatively thick or dense part of the cell (such as the nucleus) is retarded and its phase consequently shifted relative to light that has passed through an adjacent (thinner) region of the cytoplasm. Phase contrast microscopy is often used for enzyme detection. Reflection contrast microscopy

Reflection contrast microscopy is similar to phase contrast microscopy. This technique measures the shift of wavelength of the light reflected from the sample relative to that of directly emitted light. Bonnet (personal communication) made the crucial observation that with reflection contrast microscopy (Ploem, 1975; Van der Ploeg and Van D uijn, 1979; Landegent et

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al., 1985a) the DAB/peroxidase product displays bright reflection when present in extremely low amounts (i.e., low local absorption, typically A < 0.05). This property has made reflection contrast microscopy instrumental in detecting the first single copy gene by nonradioactive means (Landegent et al., 1985b; H opman et al., 1986b; Ambros et al., 1986). Studies of DAB image formation in reflection contrast microscopy have shown that best results require thin objects and low local absorbances of the DAB product (Cornelese ten Velde et al., 1988).

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Fluorescence microscopy

A fluorescence microscope contains a lamp for excitation of the fluorescent dye and a special filter which transmits a high percentage of light emitted by the fluorescent dye. Usually antifading reagents have to be added before analysis. Fluorescence microscopy is of great value for nonradioactive in situ hybridization. It is highly sensitive. Furthermore, it can be used to excite three different immunofluorophores with spectrally well-separated emissions (which allows multiple detection). Also the option of using highly sensitive image acquisition systems and the relative ease of quantitation of fluorescence signals adds to its attractiveness. Digital imaging microscopy

Digital imaging microscopes can detect signals that cannot be seen with conventional microscopes. Also, image processing technology provides enhancement of signal-tonoise ratios as well as measurement of quantitative data. The most sensitive system to date, the cooled CCD (charged coupled device) camera, counts emitted photons with a high efficiency over a broad spectral range of wavelengths, and thus is the instrument of choice for two-dimensional analysis. For three-dimensional analysis, confocal laser scanning microscopy is used. Electron microscopy

The resolution of microscopy is enhanced when electrons instead of light are used, since electrons have a much shorter wavelength (0.004 nm). The practical resolving power of most modern electron microscopes is 0.1 nm. With the electron microscope, the fine structure of a cell can be resolved. Such analysis requires special preparative procedures which are described in detail in Chapter 5.

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Flow cytometry The enormous speed with which fluorescence of individual cells can be measured with a flow cytometer has many advantages for quantitating in situ hybridization

signals. Procedures for fluorescence in situ hybridization in suspension have been described (Trask et al., 1985) and further developments in this field will be very important.

Flow diagram for in situ hybridization

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Preparation of slides/ coverslips – e.g., gelatin or poly-lysine treatment of slides – siliconization of coverslips Fixation of material on slide – by precipitation (e.g., ethanol) – by cross-linkage (e.g., formaldehyde) Pretreatment of specimen (optional) a) Treatments to prevent background staining – endogenous enzyme inactivation – RNase-treatment b) Permeabilization – diluted acids – detergent/alcohol – proteases Prehybridization (optional) Incubation of specimen with a pre-hybridization solution (= hybridization solution minus probe) is performed at the same temperature as hybridization Denaturation of probe and target – pH or heat – simultaneous or separate denaturation of probe and target (if double stranded) Hybridization Components of the solution are mainly: – Denhardt’s Mix (Ficoll, BSA, PVP) – heterologous nucleic acids (e.g., herring sperm DNA/ tRNA/competitor DNA) – sodium phosphate, EDTA, SDS, salt – formamide – dextran sulfate

Probe a) Choice of the probe – ds: DNA, cDNA – ss: RNA, oligonucleotide ss-DNA b) Preparation of the probe – DNA: fragment isolation (optional) – cDNA: cloning – RNA: cloning in transcription vectors – oligonucleotides: chemical synthesis c) Labeling of the probe – ds DNA: random primed DNA labeling, nick translation, PCR – RNA: In vitro transcription, RT-PCR – oligonucleotides: endlabeling or tailing Determination of hybridization conditions, e.g., – determination of hybridization temperature, pH, use of formamide, salt concentration – composition of hybridization solution – probe concentration

Post-hybridization steps – treatment with single strand specific nuclease (optional) – stringency washes

Fluorescence microscopy for probes directly labeled with a fluorochrome

Immunological detection – blocking step – antibody incubation – colorimetric substrate or fluorescence microscopy – counterstaining – mounting Microscopy – microscopic analysis of results and documentation Evaluation

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Determination of hybridization specificity (controls) – nuclease treatment – use of multiple probes – use of heterologous nucleic acid/vectors

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References Ambros, P. F.; Matzke, M. A.; Matzke, A. J. M. (1986) Detection of a 17 kb unique (T-DNA) in plant chromosomes by in situ hybridization. Chromosoma 94, 11–16. Burns, J.; Chan, V. T. W.; Jonasson, J. A.; Fleming, K. A.; Taylor, S.; McGee, J. O. D. (1985) Sensitive system for visualizing biotinylated DNA probes hybridized in situ: rapid sex determination of intact cells. J. Clin. Pathol. 38, 1085–1092. Cornelese ten Velde, I.; Bonnet, J.; Tanke, H. J.; Ploem, J. S. (1988) Reflection contrast microscopy. Visualization of (peroxidase generated) diaminobenzidine polymer products and its underlying optical phenomena. Histochemistry 89, 141–150. Cremers, A. F. M.; Jansen in de Wal, N.; Wiegant, J.; Dirks, R. W.; Weisbeek, P.; Van der Ploeg, M.; Landegent, J. E. (1987) Nonradioactive in situ hybridization. A comparison of several immunocytochemical detection systems using reflection contrast microscopy and electron microscopy. Histochemistry 86, 609–615. Dauwerse, J. G.; Wiegant, J.; Raap, A. K.; Breuning, M. H.; Van Ommen, G. J. B. (1992) Multiple colors by fluorescence in situ hybridization using ratiolabeled DNA probes create a molecular karyotype. Hum. Molec. Genet. 1, 593–598. De Jong, A. S. H.; Van Kessel-Van Vark, M.; Raap, A. K. (1985) Sensitivity of various visualization methods for peroxidase and alkaline phosphatase activity in immunoenzyme histochemistry. Histochem. J. 17, 1119–1130. Gallyas, F.; Gorcs, T.; Merchenthaler, I. (1982) High grade intensification of the end-product of the diaminobenzidine reaction for peroxidase histochemistry. J. Histochem. Cytochem. 30, 183–184. Garson, J. A.; Van den Berghe, J. A.; Kemshead, J. T. (1987) Novel non-isotopic in situ hybridization technique detects small (1 kb) unique sequences in routinely G-banded human chromosomes: fine mapping of N-myc and b-NGF genes. Nucleic Acids Res. 15, 4761–4770. Graham, R. C.; Karnovsky, M. J. (1966) The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14, 291–302. Heyting, C.; Kroes, W. G. M.; Kriek, E.; Meyer, I.; Slater, R. (1985) Hybridization of N-acetoxy-N-acetyl2-aminofluorene labeled RNA to Q-banded metaphase chromosomes. Acta Histochem. 77, 177–184. Hopman, A. H. N.; Wiegant, J.; Raap, A. K.; Landegent, J. E.; Van der Ploeg, M.; Van Duijn, P. (1986b) Bi-color detection of two target DNAs by nonradioactive in situ hybridization. Histochemistry 85, 1–4. Hsu, S. M.; Soban, E. (1982) Color modification of diaminobenzidine (DAB) precipitation by metallic ions and its application for double immunohistochemistry. J. Histochem. Cytochem. 30, 1079–1082.

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Landegent, J. E., Jansen in de Wal, N.; Ommen, G. J. B.; Baas, F.; De Vijlder, J. J. M.; Van Duijn, P.; Van der Ploeg, M. (1985b) Chromosomal localization of a unique gene by non-autoradiographic in situ hybridization. Nature 317, 175–177. Landegent, J. E.; Jansen in de Wal, N.; Ploem, J. S.; Van der Ploeg, M. (1985a) Sensitive detection of hybridocytochemical results by means of reflection contrast microscopy. J. Histochem. Cytochem. 33, 1241–1246.

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Nederlof, P .M.; Van der Flier, S.; Wiegant, J.; Raap, A. K.; Tanke, H. J.; Ploem, J. S.; Van der Ploeg, M. (1990) Multiple fluorescence in situ hybridization. Cytometry 11, 126–131. Nederlof, P. M.; Van der Flier, S.; Vrolijk, J.; Tanke, H. J.; Raap, A. K. (1992) Quantification of in situ hybridization signals by fluorescence digital imaging microscopy. III. Fluorescence ratio measurements of double labeled probes. Cytometry 13, 838–845. Ploem, J. S. (1975) Reflection contrast microscopy as a tool for investigation of the attachment of living cells to a glass surface. In: Van Furth, R. (Ed.) Mononuclear Phagocytes in Immunity, Infection and Pathology. Oxford: Blackwell, 405–421. Reid, T.; Baldini, A.; Rand, T. C.; Ward, D. C. (1992) Simultaneous visualization of seven different probes by in situ hybridization using combinatorial fluorescence and digital imaging microscopy. Proc. Natl. Acad. Sci. USA 89, 1388–1392. Scopsi, L.; Larsson, L. I. (1986) Increased sensitivity in peroxidase immunocytochemistry. A comparative study of a number of peroxidase visualization methods employing a model system. Histochemistry 84, 221–230. Straus, W. (1982) Imidazole increases the sensitivity of the cytochemical reaction for peroxidase with diaminobenzidine at a neutral pH. J. Histochem. Cytochem. 30, 491–493. Trask, B.; Van den Engh, G.; Landegent, J.; Jansen in de Wal, N.; Van der Ploeg, M. (1985) Detection of DNA sequences in nuclei in suspension by in situ hybridization and dual beam flow cytometry. Science 230, 1401–1403. Van der Ploeg, M.; Van Duijn, P. (1979) Reflection versus fluorescence. Histochemistry 62, 227–232. Wiegant, J.; Wiesmeijer, C. C.; Hoovers, J. M. N.; Schuuring, E.; D’Azzo, A.; Vrolijk, J.; Tanke, H. J.; Raap, A. K. (1993) Multiple and sensitive in situ hybridization with rhodamine-, fluorescein-, and coumarin-labeled DNAs. Cytogenet. Cell Genet. 63, 73–76. Wiegant, J.; Dauwerse, J. G. (1995) Multiple-colored chromosomes by fluorescence in situ hybridization. In: Verma, R. S.; Babu, A. (Eds.) Human Chromosomes: Principles and Techniques, 2nd ed. New York: McGraw-Hill, Inc.

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Procedures for In Situ H ybridization to Chromosomes , Cells, and Tissue Sections

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Procedures for In Situ Hybridization to Chromosomes, Cells, and Tissue Sections This chapter includes contributions from several leading scientists, from researchers in molecular biology to clinical pathologists, who practice in situ hybridization. Protocols are given for in situ hybridizations on widely varying substrates, e.g., chromosome spreads, chromosomes in suspension, single cells, paraffin-embedded tissue sections, ultrathin tissue sections, and whole mount preparations. H ybridization methods are described for both D N A and RN A targets.

These procedures use digoxigenin, biotin, and fluorochromes for labeling D N A, RN A and oligonucleotides. Labeling techniques include the classical methods, such as random primed D N A labeling, nick translation, and oligonucleotide tailing with terminal transferase, as well as more modern methods such as PC R and “PRIN S”, a novel and highly sensitive approach to in situ hybridization, in which the probe is labeled after its hybridization to the target nucleic acid.

Applications covered include gene mapping, gene expression, developmental biology, tumor biology, cell sorting, clinical cytogenetics, and analysis of infectious diseases.

Please note that the protocols have been optimized by members of the individual laboratories and can be varied if necessary.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections

Contents ISH to whole chromosomes In situ hybridization to human metaphase chromosomes using DIG-, biotin- or fluorochrome-labeled DNA probes and detection with fluorochrome conjugates. 62 J. W iegant, D epartm ent of C ytochem istry and C ytom etry, U niv ersity of L eiden, T he N etherlands; U . M athieu and P. L ichter, G erm an C ancer R esearch C enter, H eidelberg, G erm any; J. W ienberg and N . A rnold, I nstitute for A nthropology and H um an G enetics, U niv ersity of M unich, G erm any ; S. Popp and T. C rem er, I nstitute for H um an G enetics and A nthropology, U niv ersity of H eidelberg, G erm any ; D . C . Ward, H um an G enetics D epartm ent, Yale U niv ersity, N ew H av en, C onnecticut, U SA ; S. Joos and P. L ichter, G erm an C ancer R esearch C enter, H eidelberg, G erm any. Detection of chromosomal imbalances using DOP-PCR and comparative genomic hybridization (CGH). 72 S. Joos, B. Schütz , M . Bentz , and P. L ichter, G erm an C ancer R esearch C enter, H eidelberg, G erm any. Identification of chromosomes in metaphase spreads and interphase nuclei with PRINS Oligonucleotide Primers and DIG- or rhodamine-labeled nucleotides. 79 R . Seibl, R esearch L aboratories, Boehringer M annheim G m bH , Penz berg, G erm any. Identification of chromosomes in metaphase spreads with DIG- or fluorescein-labeled human chromosome-specific satellite DNA probes. 88 G . Sagner, R esearch L aboratories, Boehringer M annheim G m bH , Penz berg, G erm any. Fluorescence in situ hybridization of a repetitive DNA probe to human chromosomes in suspension. 94 D . C eleda, U . Bettag, and C . C rem er, I nstitute for A pplied Physics and I nstitute for H um an G enetics and A nthropology, U niv ersity of H eidelberg, G erm any. A simplified and efficient protocol for nonradioactive in situ hybridization to polytene chromosomes with a DIG-labeled DNA probe. 97 E. R . Schm idt, I nstitute for G enetics, Johannes G utenberg-U niv ersity of M ainz , G erm any.

5

Multiple-target DNA in situ hybridization with enzyme-based cytochemical detection systems. 100 E. J. M . Speel, F. C . S. R am eaek ers, and A . H . N . H opm an, D epartm ent of M olecular C ell Biology & G enetics, U niv ersity of L im burg, M aastricht, T he N etherlands. DNA in situ hybridization with an alkaline phosphatase-based fluorescent detection system. 108 G . Sagner, R esearch L aboratories, Boehringer M annheim G m bH , Penz berg, G erm any.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections

ISH to cells Combined DNA in situ hybridization and immunocytochemistry for the simultaneous detection of nucleic acid sequences, proteins, and incorporated BrdU in cell preparations. 110 E. J. M . Speel, F. C . S. R am eaek ers, and A . H . N . H opm an, D epartm ent of M olecular C ell Biology & G enetics, U niv ersity of L im burg, M aastricht, T he N etherlands.

In situ hybridization to mRNA in in vitro cultured cells with DNA probes. 116 D epartm ent of C ytochem istry and C ytom etry, U niv ersity of L eiden, T he N etherlands. Identification of single bacterial cells using DIG-labeled oligonucleotides. 119 B. Z arda, R . A m ann, and K.-H . Schleifer, I nstitute for M icrobiology, Technical U niv ersity of M unich, G erm any.

ISH to tissues Detection of HPV11 DNA in paraffin-embedded laryngeal tissue with a DIG-labeled DNA probe. 122 J. R olighed and H . L indeberg, EN T-departm ent and I nstitute for Pathology A arhus U niv ersity H ospital, D enm ark . Detection of mRNA in tissue sections using DIG-labeled RNA and oligonucleotide probes. 126 P. Kom m inoth, D iv ision of C ell and M olecular Pathology, D epartm ent of Pathology, U niv ersity of Z ürich, Sw itz erland. Detection of mRNA on paraffin embedded material of the central nervous system with DIG-labeled RNA probes. 136 H . Breitschopf and G . Suchanek , R esearch U nit for Experim ental N europathology, A ustrian A cadem y of Sciences, V ienna, A ustria.

5

RNA-RNA in situ hybridization using DIG-labeled probes: the effect of high molecular weight polyvinyl alcohol on the alkaline phosphatase indoxyl-nitroblue tetrazolium reaction. M . D e Block and D . D ebrouw er, Plant G enetic System s N .V., G ent, Belgium .

141

Detection of neuropeptide mRNAs in tissue sections using oligonucleotides tailed with fluorescein-12-dUTP or DIG-dUTP. 146 D epartm ent of C ytochem istry and C ytom etry, U niv ersity of L eiden, T he N etherlands.

In situ hybridization of DIG-labeled rRNA probes to mouse liver ultrathin sections. 148 D . Fischer, D . Weisenberger, and U . Scheer, I nstitute for Z oology I , U niv ersity of W ürz burg, G erm any. RNA in situ hybridization using DIG-labeled cRNA probes. 152 H . B. P. M . D ijk m an, S. M entz el, A . S. de Jong, and K. J. M . A ssm ann, D epartm ent of Pathology, N ijm egen U niv ersity H ospital, N ijm egen, T he N etherlands.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections

Whole mount ISH Localization of the expression of the segmentation gene hunchback in Drosophila embryos with DIG-labeled DNA probes. 158 D . Tautz , I nstitute for G enetics and M icrobiology, U niv ersity of M unich, G erm any. Detection of even-skipped transcripts in Drosophila embryos with PCR/DIG-labeled DNA probes. 162 N . Patel and C . G oodm an, C arnegie I nstitute of Washington, Em bryology D epartm ent, Baltim ore, M aryland, U SA . Whole mount fluorescence in situ hybridization (FISH) of repetitive DNA sequences on interphase nuclei of the small cruciferous plant Arabidopsis thaliana. 165 Serge Bauw ens and Patrick Van O ostv eldt, L aboratory for G enetics and L aboratory for Biochem istry and M olecular C ytology, G ent U niv ersity, G ent, Belgium .

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

In situ hybridization to human metaphase chromosomes using DIG-, biotin-, or fluorochromelabeled DNA probes and detection with fluorochrome conjugates J. Wiegant, D epartm ent of C ytochem istry and C ytom etry, L eiden U niv ersity, N etherlands.

In recent years, a number of improvements in chromosomal in situ hybridization protocols have been achieved. These have allowed a fairly high success rate for single copy gene localization as well as competition in situ hybridization. H ere we present a detailed outline of the procedure that has been successfully applied in various laboratories. The procedures have been optimized for fluorescent detection. For the second edition of this manual, we have included new procedures and new illustrations for multicolor, multitarget fluorescent detection. The procedures have also been used with slight modifications by the groups of D r. P. Lichter, D r. J. Wienberg, D r. T. C remer and D r. D . Ward. The illustrative material they have provided below demonstrates the flexibility and wide applicability of the technique.

5

At the end of the article, D r. Lichter’s group has provided a troubleshooting guide to help solve hybridization problems that may occur.

I. Pretreatment of metaphase spreads on slides (optional) ! 1 Incubate metaphase spreads with 100 µl

of 100 µg/ ml RN ase A (in 2 x SSC ) under a coverslip for 1 h at 37°C . 2 Wash slides 3 x 5 min with 2 x SSC . ! ! 3 D ehydrate slides in an ethanol series (increasing ethanol concentrations). 4 Incubate with 0.005–0.02% pepsin in ! 10 mM H C l for 10 min at 37°C . 5 Wash slides as follows: ! " 2 x 5 min with PBS. " O nce with PBS containing 50 mM MgC l2. 6 Post-fix for 10 min at room temperature ! with a solution of PBS containing 50 mM MgC l2 and 1% formaldehyde. ! 7 Wash with PBS and dehydrate (as in Step 3 above).

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II. Denaturation and hybridization For denaturation and hybridization three alternative protocols are given: A. For probes recognizing repetitive targets such as the alphoid sequences B. For probes recognizing unique sequences C. For probes or probe cocktails which contain repetitive sequences occurring throughout the genome (e.g., A lu sequences) [competition hybridization] A. For repetitive probes ! 1 U sing digoxigenin- (D IG -), biotin-, or

any fluorochrome-labeled nucleotide, label 1 µg of D N A probe according to the procedures described in C hapter 4 of this manual. 2 Precipitate the labeled probe with ! ethanol. ! 3 Prepare a probe stock solution as follows: " Resuspend the precipitated probe at a concentration of 10 ng/ µl in a solution containing 60% deionized formamide, 2 x SSC , 50 mM sodium phosphate; pH 7.0. " Incubate the tube for 15 min at 37° C with occasional vortexing until the precipitated D N A dissolves. 4 D ilute the probe from the stock to the ! desired concentration (typically 2 ng/ µl) in a solution containing 60% deionized formamide, 2 x SSC , 50 mM sodium phosphate; pH 7.0. 5 Apply 5 µl diluted probe under a cover! slip on the object slide and place the slide in an oven at 80°C for 2–4 min to denature the probe and target. N ote: Sealing w ith rubber cem ent is not m andatory. 6 To hybridize, place the slides in a moist ! chamber at 37° C overnight. ! 7 Wash the slide as follows: " 3 x 5 min at 37°C with 2 x SSC containing 60% formamide. " 1 x 5 min with the buffer to be used for immunological detection.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

B. For unique probes

! 3 Prepare a probe stock solution by

! 1 Prepare the probe stock solution as

dissolving the pellet in a solution of 50% deionized formamide, 2 x SSC , 10% dextran sulfate, 50 mM sodium phosphate; pH 7. D epending on the probe cocktail, the final concentration ranges from 10–40 ng/ µl. Mix well! Examples: Prepare a 10 ng/ µl stock solution of cosm id probes, a 20 ng/ µl stock of chrom osom e specific libraries, or a 40 ng/ µl stock of Y A C probes. 4 For hybridization, dilute the probe from ! the stock to the desired concentration in a solution of 50% deionized formamide, 2 x SSC , 10% dextran sulfate, 50 mM sodium phosphate; pH 7. Examples: For hybridiz ation, use 2–5 ng/ µl of cosm id probes, 10–20 ng/ µl of chrom osom e specific libraries, or 20–40 ng/ µl of Y A C probes. 5 D enature the probe at 75°C for 5 min, ! chill on ice, and allow annealing of the repetitive elements at 37°C for either 30 min (if C o t-1 D N A is used as competitor) or 2 h (if total human placenta D N A is used as competitor). 6 Prepare the chromosomes on the slide ! by performing the following steps: " To the chromosomes, add a solution of 70% formamide, 2 x SSC , 50 mM sodium phosphate; pH 7. C over with a coverslip. " Incubate in an oven at 80° C for 2–4 min to denature the chromosomes. " Remove the coverslip and quench the chromosomes in chilled 70% ethanol. " Dehydrate the slide by passing it through 90% ethanol, then 100% alcohol. " Air dry the slide, preferably on a metal plate at 37° C . ! 7 H ybridize the chromosomes overnight with 10 µl of the pre-annealed probe (from Step 5) under a sealed coverslip. 8 Wash the slide as follows: ! " 3 x 5 min at 45°C with 2 x SSC containing 50% formamide. " 3 x 5 min at 60°C with 0.1 x SSC . " 1 x 5 min with the buffer to be used in immunological detection.

described under Procedure IIA, but use a solution containing 50% deionized formamide, 2 x SSC , 10% dextran sulfate, 50 mM sodium phosphate; pH 7. 2 D ilute 2–5 µl of the probe stock solu! tion to 10 µl with a solution containing 50% deionized formamide, 2 x SSC , 10% dextran sulfate, 50 mM sodium phosphate; pH 7. Mix well! ! 3 To denature probe and target, do the following: " Apply 10 µl of the dilute hybridization solution under a coverslip on the object slide. " Seal coverslip to slide with rubber cement. " Place the slide in an oven at 80°C for 2–4 min. 4 To hybridize, place the slides in a moist ! chamber at 37° C overnight. 5 Wash the slide as follows: ! " 3 x 5 min at 45°C with 2 x SSC containing 50% formamide. " 5 x 2 min with 2 x SSC . " 1 x 5 min with the buffer used in detection. C. For probes containing repetitive elements [competitive hybridization] ! 1 U sing D IG -, biotin-, or any fluoro-

chrome-labeled nucleotides, label 1 µg of D N A probe according to the procedures described in C hapter 4 of this manual. 2 To precipitate the labeled probe, do the ! following: " Add a 50-fold excess of human C o t-1 D N A (or 500-fold excess fragmented human placenta D N A), a 50-fold excess of fragmented herring sperm D N A, a 50-fold excess of yeast tRN A, 1/ 10th volume of 3 M sodium acetate ( pH 5.6), and 2.5 volumes of cold (–20°C ) ethanol. " Incubate for 30 min on ice. " C entrifuge for 30 min at 4°C . " D iscard the supernatant and dry the pellet.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

III. Single color fluorescent detection with immunological amplification A1. Biotin-labeled probe, low sensitivity ! 1 Wash slides briefly with TN T buffer

[100 mM Tris-H C l (pH 7.5), 150 mM N aC l, 0.05% Tween ® 20]. 2 Incubate for 30 min at 37°C with TN B ! buffer [100 mM Tris-H C l (pH 7.5), 150 mM N aC l, 0.5% blocking reagent]. ! 3 Incubate for 30 min at 37°C with the proper dilution (typically 10 µg/ ml) of a fluorescently labeled streptavidin conjugate in TN B. 4 Wash the slides (3 x 5 min) with TN T. ! 5 Prepare the slides for viewing by per! forming the following steps: " D ehydrate through an ethanol series (70% , then 90% , then 100% ethanol; 5 min each). " Air dry. " Stain with the appropriate D N A counterstain. " Embed in Vectashield (Vector). 6 View the slides by fluorescence micro! scopy, using appropriate filters. A2. Biotin-labeled probe, high sensitivity ! 1 Wash slides briefly with TN T buffer

[100 mM Tris-H C l (pH 7.5), 150 mM N aC l, 0.05% Tween ® 20]. 2 Incubate for 30 min at 37°C with TN B ! buffer [100 mM Tris-H C l (pH 7.5), 150 mM N aC l, 0.5% blocking reagent]. ! 3 Incubate for 30 min at 37°C with the proper dilution (typically 10 µg/ ml) of a fluorescently labeled streptavidin conjugate in TN B. 4 Wash the slides (3 x 5 min) with TN T. ! 5 Incubate the slides for 30 min at 37°C ! with the proper dilution of biotinylated goat anti-streptavidin (5 µg/ ml) in TN B. 6 Repeat the washes as in Step 4. ! ! 7 Repeat Step 3. 8 Repeat the washes as in Step 4. ! ! 9 Prepare the slides for viewing as in the biotin low sensitivity procedure (Procedure IIIA1), Step 5.

5

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B1. Digoxigenin-labeled probe, low sensitivity ! 1 Wash slides briefly with TN T buffer

[100 mM Tris-H C l (pH 7.5), 150 mM N aC l, 0.05% Tween 20]. 2 Incubate for 30 min at 37°C with TN B ! buffer [100 mM Tris-H C l (pH 7.5), 150 mM N aC l, 0.5% blocking reagent]. ! 3 Incubate for 30 min at 37°C with the proper dilution (typically 2 µg/ ml, in TN B) of a fluorescently labeled sheep anti-D IG antibody. 4 Wash the slides (3 x 5 min) with TN T. ! 5 Prepare the slides for viewing as in the ! biotin low sensitivity procedure (Procedure IIIA1), Step 5. B2. Digoxigenin-labeled probe, high sensitivity ! 1 Wash slides briefly with TN T buffer

[100 mM Tris-H C l (pH 7.5), 150 mM N aC l, 0.05% Tween ® 20]. 2 Pipette 100 µl of TN B buffer [100 mM ! Tris-H C l (pH 7.5), 150 mM N aC l, 0.5% blocking reagent] onto each slide and cover with a 24 x 50 mm coverslip. Incubate for 30 min at 37°C in a moist chamber (1 L beaker containing moistened tissues, covered with aluminum foil). ! 3 Immerse the slides for 5 min in TN T to loosen the coverslips. 4 Prepare fresh working solutions of three ! antibodies: " Antibody 1 working solution: mouse monoclonal anti-D IG , 0.5 µg/ ml in TN B. " Antibody 2 working solution: D IG conjugated sheep anti-mouse Ig, 2 µg/ ml in TN B. " Antibody 3 working solution: fluorescein- or rhodamine-conjugated sheep anti-D IG , 2 µg/ ml in TN B. N ote: T he three antibodies used in this procedure are also av ailable in the Fluorescent A ntibody Enhancer Set for D I G D etection. 5 Pipette 100 µl of antibody 1 working solu! tion onto each slide and cover with a 24 x50mm coverslip.Incubatefor 30min at 37°C in a moist chamber.

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6 Wash the slides (3 x 5 min) at room !

temperature with TN T.

! 7 Pipette 100 µl of antibody 2 working

solution onto each slide and add a 24 x 50 mm coverslip. Incubate for 30 min at 37° C in a moist chamber. 8 Repeat the washes as in Step 6. ! ! 9 Pipette 100 µl of antibody 3 working solution onto each slide and add a 24 x 50 mm coverslip. Incubate for 30 min at 37° C in a moist chamber. N ote: For ev en higher sensitiv ity, repeat Steps 5–8 before perform ing the incubation w ith antibody 3 (Step 9). 10 Repeat the washes as in Step 6. ! 11 Prepare the slides for viewing as in the ! biotin low sensitivity procedure (Procedure IIIA1), Step 5. C1. Fluorescently labeled probes (fluorescein, coumarin, CY3, rhodamine, Texas Red™), low sensitivity

After the posthybridization washes (Procedure II), prepare the slides for viewing as in the biotin low sensitivity procedure (Procedure IIIA1), Step 5. C2. Fluorescein-labeled probes, high sensitivity ! 1 Wash slides briefly with TN T buffer

[100 mM Tris-H C l (pH 7.5), 150 mM N aC l, 0.05% Tween ® 20]. 2 Incubate for 30 min at 37°C with TN B ! buffer [100 mM Tris-H C l (pH 7.5), 150 mM N aC l, 0.5% blocking reagent]. ! 3 Incubate for 30 min at 37°C with the proper dilution of an anti-fluorescein antibody (either polyclonal or monoclonal) in TN B. 4 Wash the slides (3 x 5 min) with TN T. ! 5 Depending on the antibody used in Step 3, ! incubate with the proper dilution of a fluorescently labeled rabbit anti-mouse or goat anti-rabbit antibody in TN B for 30 min at 37° C . 6 Repeat the washes as in Step 4. ! ! 7 Prepare the slides for viewing as in the biotin low sensitivity procedure (Procedure IIIA1), Step 5.

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IV. Multicolor fluorescence in situ hybridization (Multicolor FISH) For multicolor FISH , several sets of probes with different labels must be hybridized simultaneously to the target and visualized with combinations of antibodies. Table 1 lists an antibody-probe matrix which allows a triple color detection of three differently labeled chromosome specific libraries (so-called chromosome painting probes). Incubation

Probe 1 Biotin

Probe 2 D igoxigenin

Probe 3 Fluorescein

1

StreptavidinTexas Red™

Mouse antiD IG antibody

Rabbit antifluorescein antibody

2

Biotin-labeled anti-streptavidin

Sheep anti-mouse FITC -labeled goat antibody anti-rabbit antibody

3

StreptavidinTexas Red™

AMC A-labeled sheep anti-D IG antibody

N ote:Be careful that the selected antibodies do not cross-react. Outline of protocol for multicolor FISH ! 1 Simultaneously hybridize all probes

listed in Table 1 to the target.

5

2 For each of the 3 detection incubations, !

perform the following steps: " Prepare the antibodies needed in the incubation (as listed in Table 1) at the proper dilution in the correct buffer. N ote: U se the procedures in Section I I I abov e as a guideline. " Mix all antibodies needed in the incubation and incubate simultaneously with the slide. " Perform blocking and washing steps as in Section III above. ! 3 Repeat Step 2 until all incubations are complete. 4 D o not counterstain the slides, but ! dehydrate, air dry, and mount the slides as in the biotin low sensitivity procedure (Procedure IIIA1), Step 5.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

V. Results obtained with human metaphase chromosome spreads The following examples (from our laboratory and other laboratories) show different applications of the procedures described in this section . A. Multicolor FISH of human lymphocyte metaphase and interphase cells from the D epartm ent of C ytochem istry and C ytom etry, L eiden U niv ersity, N etherlands

The techniques described above allow the detection of two, three, or many different probes simultaneously (Figures 1–4). Figure 1: FISH of CY3-labeled " satelliteIII probepUC1.77(chromosome 1) to human lymphocyte metaphase and interphase cells. The slides were counterstained with YOYO-1. The photomicrograph was taken with a double band-pass fluorescence filter to allow simultaneous visualization of fluorescein and Texas Red™.

5

N ick translation was used to label a chromosome 17-specific alphoid DN A (with digoxigenin) and a chromosome 18-specific alphoid DN A (with biotin). Both the digoxigenin-labeled and the biotin-labeled probes were hybridized to a chromosome sample from a leukemia patient. The procedures described above for repetitive DN A (Procedure IIA of this article) were used for slide preparation, labeling, and hybridization, except the following minor modifications were necessary for alphoid DN A: ! 1 After nick translation labeling, concen-

trate the probe (approx. 10–30 ng D N A) by drying the D N A directly in the tube. N ote: Ethanol precipitation is not necessary. 2 D issolve the dried D N A pellet in 60% !

formamide, 2 x SSC . N ote: D o not use dextran sulfate in the resuspension buffer. O m ission of dextran sulfate reduces the chance of cross-hybridiz ation.

Figure 2: Triple color FISH of " coumarin-labeled satellite III probe pUC1.77 (chromosome 1), fluorescein-labeled alphoid probe pBamX5 (chromosome X), and rhodamine-labeled alphoid probe p17H8 (chromosome 17) to human lymphocyte metaphase and interphase cells. The photomicrograph was taken by superimposing the coumarin, fluorescein, and rhodamine images.

! 3 After hybridization, use the following

washes: " 3 x 5 min at 42°C with 60% formamide, 2 x SSC ; pH 7. N ote: C om pared to the standard protocol the concentration of form am ide is increased up to 60% .

#

Figure 3: Double color FISH of a biotin-labeled chromosome1-specificlibraryanda digoxigeninlabeled chromosome 4-specific library to human lymphocyte metaphase and interphase cells. The libraries were visualized by a combined immunocytochemical reaction using fluoresceinlabeled anti-digoxigenin and Texas Red™-labeled streptavidin. The photomicrograph was taken with a double band-pass fluorescence filter to allow simultaneous visualization of fluorescein and Texas

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B. Detection of a trisomy in a leukemia patient in interphase nuclei using double in situ hybridization from U . M athieu and D r. P. L ichter, G erm an C ancer R esearch C enter, H eidelberg, G erm any.

#

Figure 4: Twelve color FISH of 12 different ratiolabeled chromosome-specific libraries to human lymphocyte metaphase and interphase cells. For details of the experiment, see Dauwerse et al. (1992). The photomicrograph was created by superimposing the coumarin image and the image obtained with a double band-pass fluorescence filter (to allow simultaneous visualization of fluorescein and Texas Red™).

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

Figure 5 shows the result of the hybridization. The digoxigenin-labeled probe was visualized with anti-DIG-rhodamine; the biotin-labeled probe, with avidin-FITC.

D. Interphase nuclei of a patient with Ph1-positive ALL(acute lymphoblastic leukemia) from M . Bentz , P. L ichter, H . D öhner and G . C abot.

The Philadelphia chromosome (Ph1) is the derivative of a translocation between chromosome 9 and chromosome 22 [(t9;22) (q34;q11)]. Figure 7 shows the use of two probes, one specific for chromosome 9 and the other specific for chromosome 22, to detect Ph1. $ Figure 7: Double color FISH to #

Figure 5: Double color FISH of a digoxigeninlabeled chromosome 17-specific probe and a biotin-labeled chromosome 18-specific probe to a human chromosome preparation containing a trisomy. Each alphoid DNA probe was specific for the centromere of its target chromosome. In the metaphase and in the interphase, the probes detected three signals (green) from chromosome 18 and two signals (red) from chromosome 17. DAPI was used for counterstaining.

C. Detection of a trisomy and a tetrasomy in interphase nuclei of a leukemia patient with two different cosmid probes from U . M athieu and D r. P. L ichter

A digoxigenin-labeled cosmid probe specific for chromosome 14 and a biotin-labeled cosmid probe specific for chromosome 21 were used to detect polyploidy in a leukemia patient (Figure 6). Probes were labeled by nick translation and visualized with either anti-D IG -rhodamine (chromosome 14-specific probe) or avidin-FITC (chromosome 21-specific probe).

#

Figure 6: Double color FISH of a digoxigeninlabeled chromosome 14-specific cosmid probe and a biotin-labeled chromosome 21-specific cosmid probe to a human chromosome preparation of a leukemia patient containing a trisomy and a tetrasomy. In the interphase nuclei, the cosmid DNA probes detected three copies (red) of chromosome 14 and four copies (green) of chromosome 21. DAPI was used for

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E. Chromosomal in situ suppression (CISS) hybridization of the human DNA library specific for chromosome 2 to chromosomes of Gorilla gorilla from D r. J. Wienberg and D r. N . A rnold, I nstitute for A nthropology and H um an G enetics, U niv ersity of M unich, G erm any.

The hybridization pattern (Figure 8) obtained with specific D N A probe sets (Weinberg et al., 1990) confirms the assumed fusion of two submetacentric chromosomes during human evolution.

detect the Philadelphia chromosome (Ph1) in a patient with ALL. A cosmid probe specific for chromosome 9 and a YAC probe specific for chromosome 22 were hybridized to interphase nuclei (Bentz et al., 1994). These two probes detected two red (rhodamine) signals from chromosomes 22, one green (FITC) signal from chromosome 9 and one yellow signal caused by the overlapping of red (chromosome 22) and green (chromosome 9) signals on Ph1. DAPI was used for counterstaining. Reprinted from Bentz et al. (1994) Detection of Chimeric BCR–ABL genes on Bone Marrow Samples and Blood Smears in Chronic Myeloid and Acute Lymphoblastic Leukemia by In Situ Hybridization, Blood 83 (7); 1922–1928 with permission of the Journal Permissions Department, W. B. Saunder’s Company.

#

Figure 8: FISHof a chromosome 2-specific library to chromosomes of Gorilla gorilla. Standard chromosome preparations wereobtainedfromEBVtransformed lymphoblastoid cell lines. DNAprobes were from flow-sorted human chromosomes cloned to phages or plasmids. Probes were labeled with digoxigenin by nick translation. Detection was performed with monoclonal mouse anti-DIGantibody, rabbit anti-mouse-TRITC, and goat anti-rabbit-TRITC.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

F. Illustration of a chromosomal translocation from a patient with chronic myeloid leukemia from S. Popp and D r. T. C rem er, I nstitute for H um an G enetics and A nthropology, U niv ersity of H eidelberg, G erm any.

The chromosomal translocation was detected by using library probes from sorted chromosomes 9 and 22 (Figure 9). In Panel a, the chromosome 22-specific probe has



clearly painted the normal chromosome 22 (bottom), the Philadelphia chromosome (triangle) and the translocated material 22q11 → 22qter contained in the derivative chromosome 9 (arrow). In Panel b, the chromosome 9-specific probe has stained the normal chromosome 9 blue except for the centromeric region, while in the derivative chromosome 9, the region 9pter → 9q34 is also painted. The arrow points to the breakpoint on chromosome 9.



#

a

b

#

Figure 9: Two-color chromosomal in situ suppression (CISS)-hybridization of a metaphase spread obtained from a patient with chronic myeloid leukemia 46, XY, t(9;22) (q34;q11). In Panel a, the probe was a digoxigenin-labeled chromosome 22-specific library, which was detected with a mouse anti-DIG antibody and a FITC-conjugated sheep anti-mouse antibody. The same metaphase spread was simultaneously painted (Panel b) with a biotin-labeled chromosome 9-specific library, which was detected with avidin conjugated to the fluorochrome AMCA (aminocoumarin). Chromosomes were counterstained with propidium iodide. Chromosomal analysis using GTG-banded chromosomes was kindly performed by R. Becher, Westdeutsches Tumorzentrum, Essen. Figure 10: FISH of an ATPase" specific probe and a probe containing L1-repetitive sequences to female mouse chromosomes. Yellow color (banding) is due to the biotin-labeled probe containing L1-repetitive sequences, which was detected with FITC. Red „ dots“ indicate a signal from each of the four (look closely) chromatids obtained with the digoxigenin-labeled ATPasespecific probe, which was detected with anti-DIG Fab fragments (from sheep) and Texas Red™-conjugated anti-sheep Ig Fab. The blue color is the DAPI counterstain.

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G. Digitized images of female mouse chromosomesvisualizedwitha Zeiss epifluorescent microscope equipped with a cooled CCD camera from D r. D . C . Ward, H um an G enetics D epartm ent, Yale U niv ersity, N ew H av en, C T, U SA .

In Figure 10, a probe specific for N a+/ K + ATPase alpha subunits and a probe containing an L1-repetitive sequence were hybridized to female mouse chromosomes.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

Reagents available from Boehringer Mannheim for these procedures Reagent

Description

Cat. No.

Pack size

DIG-Nick Translation Mix* for in situ probes

5x conc. stabilized reaction buffer in 50% glycerol (v/v) and DNAPolymerase I, DNase I, 0.25 mMdATP, 0.25 mMdCTP, 0.25 mMdGTP, 0.17 mM dTTP and 0.08 mM DIG-11-dUTP.

1745 816

160 µl (40 labeling reactions)

Biotin-Nick Translation Mix* for in situ probes

5x conc. stabilized reaction buffer in 50% glycerol (v/v) and DNAPolymerase I, DNase I, 0.25 mM dATP, 0.25 mM dCTP, 0.25 mM dGTP, 0.17 mM dTTP and 0.08 mM biotin-16-dUTP.

1745 824

160 µl (40 labeling reactions)

Nick Translation Mix* for in situ probes

5x conc. stabilized reaction buffer in 50% glycerol, DNA Polymerase I and DNase I.

1745 808

200 µl

dNTP Set

Set of dATP, dCTP, dGTP, dTTP, 100 mM solutions, lithium salts.

1 277 049

4x10 µmol (100 µl)

Fluorescein-12-dUTP

Tetralithium salt, 1 mM solution

1 373 242

25 nmol (25 µl)

Tetramethylrhodamine-6-dUTP

Tetralithium salt, 1 mM solution

1 534 378

25 nmol (25 µl)

AMCA-6-dUTP

Tetralithium salt, 1 mM solution

1 534 386

25 nmol (25 µl)

RNase A

Pyrimidine specific endoribonuclease which acts on single-stranded RNA.

109 142 109 169

25 mg 100 mg

Pepsin

Aspartic endopeptidase with broad specificity.

108 057 1 693 387

1g 5g

DNA, COT-1, human

COT-1 DNA is used in chromosomal in situ suppression [CISS] hybridization.

1 581 074

500 µg (500 µl)

DNA, MB-grade from fish sperm

The ready-to-use solution is directly added to the hybridization mix.

1 467 140

500 mg (50 ml)

tRNA from baker’s yeast

Lyophilizate, 1 mg of dry substance corresponds to 16 A260 units.

109 495 109 509

100 mg 500 mg

Tween® 20

Aqueous solution, 10% (w/v)

1 332 465

5x10 ml

Tris

Powder

127 434 708 968 708 976

100 g 500 g 1 kg

Blocking Reagent, for nucleic acid hybridization

Powder

1 096 176

50 g

Anti-Digoxigenin, clone 1.71.256, mouse IgG1, !

For the detection of digoxigenin-labeled compounds

1 333 062

100 µg

Anti-DigoxigeninFluorescein, Fab fragments from sheep

For the detection of digoxigenin-labeled compounds

1 207 741

200 µg

Anti-DigoxigeninRhodamine, Fab fragments from sheep

For the detection of digoxigenin-labeled compounds

1 207 750

200 µg

Anti-Mouse Ig-Digoxigenin, F(ab)2 fragment

Antibody for triple color FISH

1 214 624

200 µg

Streptavidin-AMCA

For the detection of biotin-labeled compounds.

1 428 578

1 mg

Streptavidin-Fluorescein For the detection of biotin-labeled compounds.

1 055 097

1 mg

236 276

10 mg

DAPI Fluorescence dye for staining of 4',6-Diamidine-2'-Phenyl- chromosomes indole Dihydrochloride

CONTENTS

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5

*This product or the use of this product may be covered by one or more patents of Boehringer Mannheim GmbH, including the following: EP patent 0 649 909 (application pending).

69

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

VI. Troubleshooting guide for in situ hybridization on chromosome spreads from Stefan Joos and Peter L ichter, G erm an C ancer R esearch C enter, H eidelberg, G erm any. U se the following tips to diagnose and correct commonly occurring problems in the FISH protocols described above. A. If no signal is observed, then: ! 1 Amplify signal. 2 C heck probe labeling by performing the !

following dot blot assay: " Spot serial dilutions of labeled control. " Spot serial dilutions of labeled sample. " C ompare intensities of the spots. N ote: For the detailed procedure on how to estim ate the labeling efficiency please refer to C hapter 4, I X , page 51. ! 3 After labeling the probe according to

Steps 1–4 of the nick translation procedure (Procedure III) in C hapter 4 of this manual, check the size of the labeled probe molecules as follows: " To a 5–10 µl aliquot of probe, add gel loading buffer and denature the probe by incubating it in a boiling water bath for 3 min, then cool it on ice for 3 min. N ote:Keep the rem ainder of the probe sam ple on ice w hile running the gel.

5

" Load the aliquot on a standard 1–2%

agarose minigel along with a suitable size marker. " Q uickly run the gel (e.g. 15 volts per cm for 30 min) to avoid renaturation of the probe in the gel. " Visualize D N A in the gel, e.g. by staining gel in 0.5 µg/ ml ethidium bromide, and take photograph during U V illumination. N ote: T he probe m olecules w ill be v isible as a sm ear. T he sm ear should contain only fragm ents sm aller than 500 nucleotides (nt) and larger than 100 nt. A peak intensity at 250– 300 nt seem s optim al. " D epending on the size of the probe

molecules, do one of the following: I f the D N A is between 100–500 nt, proceed with the ED TA inactivation of the reaction mixture (as in Step 7 of the nick translation procedure, C hapter 4).

70

" I f the probe is larger than 500 nt,

add more D N ase I (roughly about 1–10 ng) to the probe sample kept on ice, then incubate longer at 15°C . N ote: U sually higher concentrations of D N ase m ust be added for an additional 30 m in incubation. A fter the incubation, analyz e the probe on a gel as abov e. " I f the D N A is almost or completely

undigested, purify the probe and repeat the labeling step. I f part of the D N A is smaller than 100 nt, repeat the labeling step using less D N ase I. I f all the D N A is smaller than 100 nt, repurify the probe (to remove any possible contaminating D N ase) and repeat the labeling step. 4 C heck whether the fluorochromes have ! separated from the detecting molecules by passing the solution of fluorochromeconjugated molecules through a Q uick Spin™ column (G -50). Then determine the state of the molecules: " I f color remains in flow through, fluorochromes are still conjugated. " If color remains in the upper part of the column, fluorochromes have separated from the detecting molecules. 5 Modify ! chromosome denaturation procedure (Procedure II in this article) by either varying the time and temperature of the denaturation step or by further pepsin digestion according to the following protocol: N ote: D igestion w ith pepsin can significantly im prov e probe penetration, but ov erdigestion can also occur. Pepsin treatm ent is often useful w hen specim en preparations are suboptim al, e.g., in m any clinical sam ples. " Mark the area of interest on the back

of the slide using a diamond stylus.

" Wash slide in 2 x SSC at 37° C for

5 min (in a C oplin jar).

" Apply 120 µl RN ase A solution

(100 µg/ ml RN ase A in 2 x SSC ) to slide, cover with larger coverslip (e.g. 22 x 50 mm), and incubate for 1 h at 37°C in a moist chamber. " Wash slide in 2 x SSC (or PBS) for 3 x 5 min (C oplin jar). " Wash slide in prewarmed PBS for 5 min at 37° C .

CONTENTS

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

" Incubate slide in pepsin solution

(10 mg pepsin in 100 ml 10 mM H C l) for 10 min at 37° C (in a water bath). " Wash slide in PBS for 5 min at room temperature (RT). " Incubate slide for 10 min at RT in a solution of 1% acid-free formaldehyde, PBS, 50 mM MgC l2. " Wash slide again in PBS for 5 min at RT. " D ehydrate slide in a series of ethanol baths (70% , then 90% , then 100% ; ≥ 5 min each) and air dry. 6 C heck quality of the formamide. ! D eionize formamide (several batches from several sources should be tested) using ion exchange resin (e.g. D onex XG 8). ! 7 C heck microscope. Be sure that mercury (H g2) lamp of the microscope is in good condition and well adjusted. O ld lamps don’t have good excitation properties. 8 Perform control experiments with ! alphoid D N A probes.

D. If there is a starry background fluorescence, then: %

Possible cause

Remedy

Agglutination of fluorochromes coupled to antibodies

Perform a control experiment in the absence of probe to see if you get a non-specific signal from the fluorochrome-conjugated antibody alone. I f you do, spin the detection solution briefly and take only the supernatant. Alternatively pass the detection solution through a G -50 Q uick Spin column to remove the agglutinates.

Labeled probe molecules are too long

C heck the size of the probe as described in Step A3 above and label again.

E. If there is a general strong staining of chromosomes and nuclei, then: " C heck the size of the labeled probe (as in

Step A3 above). It is most likely too small. " I f the probe is too small, repurify (to remove any contaminating D N ase) and relabel the probe.

B. If the hybridization signal fades, then: ! 1 C hange to a different batch of fluoro-

chrome-conjugated antibody. with a secondary and/ or tertiary antibody to increase the signal. N ote: Be aw are of the back ground, w hich w ill also be am plified. Before amplification, remove embedding medium by washing (2 x SSC, 37°–42°C, 4 x 10 min). ! 3 Try different antifading solutions [e.g. 1,4-diazobicyclo-(2,2,2)-octane (DAPCO ) or Vectastain (Vector)]. 2 Amplify !

C. If there is a milky background fluorescence, then: %

References Bentz, M.; Cabot, G.; Moos, M.; Speicher, M. R., Ganser, A.; Lichter, P.; Döhner, H. (1994) Detection of chimeric BCR-ABL genes on bone marrow samples and blood smears in chronic myeloid and acute lymphoblastic leukemia by in situ hybridization. Blood 83, 1922–1928. Dauwerse, J. G.; Wiegant, J.; Raap, A. K.; Breuning, M. H.; Van Ommen, G. J. B. (1992) Multiple colors by fluorescence in situ hybridization using ratiolabeled DNA probes create a molecular karyotype. Hum. Mol. Genet. 1, 593–598. Wienberg, J.; Jauch, A.; Stanyon, R.; Cremer, T. (1990) Molecular cytotaxonomy of primates by chromosomal in situ suppression hybridization. Genomics 8, 347–350.

Possible cause

Remedy

Inadequate quality of chromosome preparation

Treat with pepsin to remove cell debris (see protocol in Step A5 above).

Insufficient blocking

Try to block with different reagents such as 3% BSA, human serum, or 3–5% dry milk.

D irty glass slides

Wash the slide with ethanol and rinse with water before spreading chromosomes.

Bad quality of embedding medium

C hange embedding medium.

CONTENTS

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5

71

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

Detection of chromosomal imbalances using DOPPCR and comparative genomic hybridization (CGH) Stefan Joos, Barbara Schütz , M artin Bentz , and Peter L ichter, G erm an C ancer R esearch C enter, H eidelberg, G erm any.

Recently, a new approach of fluorescence in situ hybridization was introduced, called “comparative genomic hybridization” (CGH ) (Kallioniemi et al., 1992), which allows the comprehensive analysis of chromosomal imbalances in entire genomes (Du Manoir et al., 1993; Joos et al., 1993; Kallioniemi et al., 1992; Kallioniemi et al., 1993). The principle of CGH is shown in Figure 1. The genomic DN A from cell populations to be tested, such as fresh or paraffin-embedded tumor tissue, is labeled with modified nucleotides (e.g., biotinylated dUTP) and used as a probe for in situ hybridization to normal metaphase chromosomes. This probe is called “test DN A”. As an total genomic DNA from cells to analyze (test DNA) labeled with biotin

total genomic DNA from normal cells (control DNA) labeled with digoxigenin

co-hybridization (normal chromosomes)

detection

5

DNA from:

results in signals on normal chromosomes (partial karyotype) signals from hybridized test DNA

signals from co-hybridized control DNA

cells with amplified DNA or polysomies cells with deletions or monosomies

Figure 1: Schematic illustration of “comparative genomic hybridization” (CGH) for chromosome analysis. For explanation, see text.

72

internal control, genomic DN A derived from cells with a normal karyotype is differentially labeled and hybridized simultaneously (“control DN A”). For detection of the hybridized test and control DN A, different fluorochromes are used, and each is visualized by epifluorescence microscopy with selective filters. H ybridization with genomic DN A results in a general staining of all chromosomes. H owever, if the tissue analyzed harbors additional chromosomal material (e.g., a trisomy, amplifications etc.), hybridization reveals higher signal intensities at the corresponding target regions of the hybridized chromosomes (Figure 1). Correspondingly, deletions in the test sample are visible as lower signal intensities (Figure 1).

By comparing the hybridization patterns of test and control DN A, the changes in signal intensities caused by imbalances within the analyzed tissue can be identified. Thus, C G H provides a comprehensive picture of all chromosomal gains and losses within a single experiment. In contrast to banding analysis, C G H requires no preparation of metaphase chromosomes from the cells to be analyzed (which in certain tumor types may be difficult or even impossible). In many cases, only small amounts of tumor tissue are available for cytogenetic analysis. Therefore it is advantageous to combine C G H with universal PC R protocols (as shown in Figures 4 and 5 under “Results”) (Speicher et al., 1993). Both sequence independent amplification (“SIA”) (Bohlander et al., 1992) and “D O P-PC R” (degenerate oligonucleotide primer PC R) (Telenius et al., 1992) can be combined with C G H . As is shown in Figure 2, the D O P primer contains a central cassette of 6 degenerated nucleotides and is flanked by specific sequences at the 3' end (6 nucleotides) and at the 5' end (10 nucleotides). The amplification procedure is divided into two steps. D uring the first step, five PC R cycles are performed at low stringency conditions (annealing temperature, T a = 30°C ). These conditions allow a frequent annealing of the D O P primer, which is primarily mediated by the short specific sequence at its 3' end and by the adjacent central cassette of degenerated nucleotides. Eventually a pool of many different D N A sequences is generated which should represent a statistically distributed subpopulation of the genomic D N A. D uring the second step (35 cycles) the annealing temperature is raised to 62°C , allowing amplification only after specific base pairing of the full length primer sequence. Therefore, all the sequences that have been synthesized in the first step are now exponentially amplified, resulting in a large amount of D N A that can be used as probe for C G H .

CONTENTS

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

Protocols for DO P-PCR and CGH analysis are described below. Amplified sequences can be detected directly by visual inspection using an epifluorescence microscope (“Results” Figures 3 and 4). H owever, the comprehensive analysis of gains and losses of single chromosomes (or parts thereof) requires digital image analysis with sophisticated software programs. For further description of the evaluation of CGH experiments as well as the different steps of the CGH protocol, see the literature (Du Manoir et al., 1995; Kallioniemi et al., 1994; Lichter et al., 1994; Lundsteen et al., 1995; Piper et al., 1995).

DOP-PCR Telenius et al., 1992 5´ CCGACTCGAGNNNNNN ATGTGG 3´

1.

Low stringency PCR (Ta = 30°C; 5 cycles) ➝ frequent priming at multiple sites 5´



I. Preparation and labeling of DOP-PCR products





! 1 Prepare a D N A template by isolating

genomic D N A from small amounts of tissue (Maniatis, Fritsch, and Sambrook, 1986). O ne mg of tissue results in about 1 µg of genomic D N A. N ote: See the literature (Kaw asak i, 1990; Speicher et al., 1993) for protocols on D N A isolation from sm aller am ounts of tissue (dow n to only a few hundred cells) or from paraffin-em bedded m aterial. 2 For each reaction, mix the following ! components in a PC R reaction tube on ice: " 5.0 µl 10 x concentrated D O P-PC R buffer (20 mM MgC l2; 500 mM KC l; 100 mM Tris-H C l, pH 8.4; 1 mg/ ml gelatin). " 5.0 µl 10 x concentrated nucleotide mix (dATP, dC TP, dG TP, dTTP, 2 mM each). " 1–10 µl genomic D N A (0.1–1 ng/ µl). " 5.0 µl 10 x concentrated D O P-PC R primer [5'-C C G AC TC G AG N N N N N N ATG TG G -3' (N =A,C ,G , or T), 20 µM]. " enough sterile water to make a final reaction volume of 50 µl. " 0.25 µlTaq D N A Polymerase (5 units/ µl) (should be added last). ! 3 Prepare a negative control by combining the same solutions as in Step 2, but omitting the template D N A. 4 O verlay reaction mixtures with 50 µl ! mineral oil.

CONTENTS

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2.

Higher stringency PCR (Ta = 62°C; 35 cycles) ➝ specific priming of pre-amplified sequences

Figure 2: Schematic illustration of DOP-PCR. For explanation, see text.

5

5 Transfer the reaction tubes to a thermo!

cycler and start the PC R. U se the following thermal profile: " Initial denaturation: 10 min at 94°C . " 5 C ycles, each consisting of: 1 min denaturation at 94°C 1.5 min annealing (low stringency) at 30° C 3 min temperature ramping, from 30° C to 72°C 3 min elongation at 72°C . " 35 C ycles, each consisting of: 1 min denaturation at 94°C 1 min annealing (high stringency) at 62° C 3 min elongation at 72° C , with the elongation time increased by 1 second every cycle [i.e., the 1st high stringency cycle has an elongation tim e of 3 m in; the 2nd, 3 m in and 1 s; the 3rd, 3 m in and 2 s, etc.] " Final elongation: 10 min at 72°C .

73

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

6 Analyze aliquots of the reaction mix!

4 C o-precipitate the probe D N As in each !

tures on an agarose gel by performing the following steps: " Mix the aliquots with gel loading buffer (0.25% bromophenol blue and 30% glycerol). " Load samples on a minigel. As a size marker use a 1 kb ladder or other suitable size standard. " Run the gel in 1x TBE buffer (89 mM Tris base, 89 mM boric acid, 2 mM ED TA; pH 8.0) for 30 min at 100 volts. ! 7 Stain the gel with ethidium bromide and photograph. In the reaction samples, you should see a smear of D N A, ranging in size from about 200 bp to 2000 bp. N o smear should be visible in the negative control. 8 C ollect the amplified D N A products ! from the reaction mix by using the H igh Pure PC R Product Purification Kit, see page 50. ! 9 Label the amplified D N A by standard nick translation procedures (Lichter and C remer; Lichter et al., 1994).

tube by adding 1/ 20 volume of 3 M sodium acetate and 2.5 volumes of 100% ethanol. Mix well and incubate at –70°C for 30 min. 5 Spin the precipitated D N A in a ! microcentrifuge at 12,000 rpm for 10 min at 4°C . D iscard the supernatant and wash the pellet with 500 µl of 70% ethanol. 6 Spin the precipitated D N A again (12,000 ! rpm, 10 min, 4°C ). D iscard the supernatant and lyophilize the pellet. ! 7 Add 6 µl deionized formamide to the lyophilizate and resuspend the probe D N A by vigorously shaking the tube for > 30 min at room temperature. 8 Add 6 µl of hybridization buffer to the ! tube containing probe D N A and again shake for > 30 min. ! 9 D enature and dehydrate normal metaphase chromosomal D N A on slides as described in the literature (Lichter and C remer; Lichter et al., 1990). 10 Prepare the probe (from Step 8) for ! hybridization by performing the following steps: " D enature probe D N A at 75°C for 5 min. " Preanneal repetitive sequences by incubating the probe at 37°C for 20–30 min. 11 Add prepared probe (from Step 10) to ! denatured chromosomal spreads (from Step 9). Proceed with in situ hybridization and probe detection as described in the literature (Lichter and C remer; Lichter et al., 1990). ! 12 Evaluate the C G H experiment with an epifluorescence microscope.

II. Comparative genomic hybridization (CGH) ! 1 Prepare slides of normal metaphase

chromosomes as described in the literature (Lichter and C remer; Lichter et al., 1990). 2 Prepare the following solutions: ! " 3 M sodium acetate, pH 5.2. " D eionized formamide (molecular biology grade): For deionization, use ion exchange resin. The conductivity of the formamide should be below 100 µSiemens. " H ybridization buffer (4 x SSC , 20% dextran sulfate): Prepare 20 x SSC . C arefully dissolve dextran sulfate in water to make a 50% dextran sulfate solution. Autoclave the dextran sulfate solution or filter it through a nitrocellulose filter. Mix 200 µl of 20 x SSC , 400 µl of 50% dextran sulfate and 400 µl of double-distilled water. Store the hybridization buffer at 4°C until you use it. ! 3 To prepare probe D N A, combine 1 µg of each labeled test and control D N A with 50–100 µg of human C o t-1 D N A.

5

74

Results The C G H procedure was used to reveal chromosomal imbalances (Figure 3) in a T-cell prolymphocytic leukemia (T-PLL). C hromosomal regions 6q, 8p, and 11qter are stained more weakly in the tumor D N A than in the control D N A, indicating deletions of all or part of the chromosomal arms in the tumor D N A. C G H also identified overrepresented chromosomal regions (6p, 8q, and 14q) which stain more heavily in the tumor than in the control.

CONTENTS

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

! Figure 3: Comparative genomic

hybridization (CGH) experiment revealing chromosomal imbalances in a T-cell prolymphocytic leukemia (T-PLL). DNA from tumor cells was labeled with biotin and detected via a FITClabeled antibody (green) whereas the control DNA was labeledwith digoxigenin and detected via a rhodamine-labeled antibody (red). For explanation, see the text. This photograph was taken by S. Joos and P. Lichter with permission of Springer Verlag, Heidelberg and New York.

! Figure 4: Detection of amplified

! Figure 5: Detection of amplified

!

The D O P-PC R and C G H procedures described in this article were used to detect amplified sequences in genomic D N A from spinal fluid of patients who have a tumor of the central nervous system (Figure 4). D O P-PC R and C G H also revealed an amplification of the c-myc oncogene (which maps to chromosomal band 8q24) in cell line H L60 (Figure 5).

!

!

!

sequences in the genomic DNA of patients with a central nervous system (CNS) tumor. Genomic DNA was isolated from a small number of cells harvested from the spinal fluid of the patients. The DNA was amplified by DOP-PCR and analyzed by CGH as described in the text. CGH reveals a strong amplification signal on both homologues of chromosome 8q (arrows). The image was photographed directly using a conventional camera on an epifluorescence microscope.

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c-myc oncogene sequences in cell line HL60. Genomic DNA (equivalent to the DNA from only 3– 4 HL60 cells) was diluted in TE buffer, amplified by DOP-PCR, and analyzed by CGHas described in the text. After CGH, the amplification signal is clearly visible on both chromosome 8 homologues (arrows). The image was captured as a gray scale image using a CCD camera (Photometrix), pseudocolored, and photographed directly from the computer screen.

75

5

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

Reagents available from Boehringer Mannheim for these procedures Product

Description

DOP-PCR Master* , **

for 25 amplification reactions and 5 control reactions

Cat. No. 1 644 963

The D O P-PCR Master contains: Reagent

Description

Available as

DOP-PCR mix, 2 x concentrated

25 units Taq DNA Polymerase/500 µl; 3 mM MgCl2; dATP, dGTP, dCTP, dTTP, 0.4 mM each; 100 mM KCl; 20 mM Tris-HCl; 0.01% (v/v) Brij ® 35; pH 8.3 (20° C)

Three vials No. 1, 3 x 500 µl

DOP-PCR primer (Sequence, 40 µM in double distilled H2O 5'- CCG ACT CGA GNN NNN NAT GTG G-3' (N = A, G, C, or T, in approximately equal proportions)

One vial No. 2, 150 µl

Double distilled H2O, PCR grade

Two vials No. 3,

2 x 1000 µl

Control DNA

Human genomic DNA from the lymphoblastoid cell line BJA, 1 ng/µl, in double distilled H2O

One vial No. 4, 50 µl

O ther reagents:

5 **This product or the use of this product may be covered by one or more patents of Boehringer Mannheim GmbH, including the following: EP patent 0 649 909 (application pending). **This product is sold under licensing arrangements with Roche Molecular Systems and The Perkin-Elmer Corporation. Purchase of this product is accompanied by a license to use it in the Polymerase Chain Reaction (PCR) process in conjunction with an Authorized Thermal Cycler. For complete license disclaimer, see inside back cover page ! + ".

76

Reagent

Description

Cat. No.

Pack size

DIG-Nick Translation Mix* for in situ probes

5x conc. stabilized reaction buffer in 50% 1745 816 glycerol (v/v) and DNAPolymerase I, DNase I, 0.25 mMdATP, 0.25 mMdCTP, 0.25 mMdGTP, 0.17 mM dTTP and 0.08 mM DIG-11-dUTP.

160 µl

Biotin-Nick Translation Mix* for in situ probes

5x conc. stabilized reaction buffer in 50% 1745 824 glycerol (v/v) and DNAPolymerase I, DNase I, 0.25 mM dATP, 0.25 mM dCTP, 0.25 mM dGTP 0.17 mM dTTP and 0.08 mM biotin-16-dUTP.

160 µl

Streptavidin-Fluorescein

1 428 578

1 mg

Anti-Digoxigenin-Rhodamine

1 207 750

200 µg

Product

Description

High Pure PCR Product Purification Kit**

Kit for 50 purifications Kit for 250 purifications

Cat. No. 1732668 1732676

The H igh Pure PCR Product Purification Kit contains: Reagent

Description

Available as

• Binding buffer, green cap • Wash buffer, blue cap

• nucleic acids binding buffer; 3 M guanidine-thiocyanate, 10 mM Tris-HCl, 5% (v/v) ethanol, pH 6.6 (25° C) • wash buffer; add 4 volumes of absolute ethanol before use! Final concentrations; 20 mM NaCl, 2 mM Tris-HCl, pH 7.5 (25° C), 80% ethanol • Elution buffer • elution buffer; 10 mM Tris-HCl, 1 mM EDTA, pH 8.5 (25° C) • High Pure filter tubes • Polypropylene tubes, containing two layers of a specially pre-treated glass fibre fleece; maximum sample volume: 700 µl • Collection tubes • 2 ml polypropylene tubes

CONTENTS

• Vial 1 • Vial 2

• Vial 3

INDEX

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

Troubleshooting CGH experiments Control to detect CGH of insufficient quality H ybridization of a D O P-PC R probe to normal human male metaphase chromosomes should show a markedly weaker staining of the X chromosome than of other chromosomes. If there is not a marked difference between the X chromosome and other chromosomes, the control indicates a defective or poor quality C G H experiment. U sually these poor hybridization results are accompanied by other hallmarks of poor quality, like a speckled, non-homogeneous staining pattern or high background fluorescence, both of which may considerably disturb the quantitative image analysis by causing high variability within the ratio profiles. Such poor hybridization results can be due to many causes. Several are listed below. Potential problems with chromosomal preparations In our experience, the most important factor for good hybridization experiments is the careful preparation of metaphase spreads. Therefore, every new batch of chromosomes needs to be tested. If good hybridization results are not obtained, discard the whole batch and start a new slide preparation procedure. N ote: A lthough im paired hybridiz ation is m ainly due to residual cell debris on the slides, proteolytic digestion of the chrom osom es w ill not lead to high quality C G H experim ents. A lthough proteolytic digestion w ill im prov e the hybridiz ation pattern on suboptim al slides, the results are considerably w orse than those obtained on optim um slides w hich hav e not been pretreated w ith protease. Potential problems with probes Two important causes for granular and non-homogeneous staining patterns are: " Probe D N A preparations may be contaminated with high amounts of protein. " The size of the labeled probe fragments after nick translation may be inappropriate. Fragments which are too large

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typically result in a starry background outside of the chromosomes. In contrast, probes which are too small produce a homogeneous background distributed over all chromosomes. Smaller probes may also lead to a homogeneously strong staining pattern in regions which are over- or under-represented in the tumor genome. Problems which may hinder evaluation of CGH In contrast to conventional FISH experiments, strong fluorescence on the chromosomes and little or no fluorescence in the areas where no chromosomes or nuclei are located, are no guarantee that a hybridization experiment will be useful for a C G H analysis. Even in cases where a bright and smooth staining is observed, other factors can severely impair evaluation of the hybridization experiment. Such impairment has two principal causes: " Insufficient suppression of repetitive sequences may lead to incorrect measurements of ratios in chromosomal regions which contain many highly variable repetitive sequences, e.g., sequences on chromosome 19. Strong staining of the heterochromatin blocks on chromosomes 1, 9, 16, and 19 can serve as an indicator for poor suppression. N ote: G ood suppression w ill lead to v ery low FI T C and rhodam ine fluorescence intensities in the heterochrom atic chrom osom e regions. C onsequently, ev en v ery sm all v ariations of fluorescence intensities m ay cause gross ratio v ariations w ithin these chrom osom al regions. T hus, they are excluded from ev aluation. Remedy: Either increase the amount of the C o t-1 D N A fraction or use longer preannealing times (the latter being less effective) to achieve adequate suppression of signals in such regions of the genome.

5

" N on-homogeneous illumination of the

optical field leads to considerable regional variations within the ratio image. Such variations make a quantitative evaluation of the C G H experiment completely impossible. Remedy: C enter the light source of the microscope carefully.

77

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

Problems inherent in sample composition Apart from the experimental factors listed above, the validity of a C G H experiment critically depends on the percentage of cells carrying chromosomal imbalances. If no gains or losses of chromosomes are detected, the sample may contain only a low proportion of tumor cells. Therefore, adequate information from the histological laboratory is crucial for any type of C G H analysis.

References Bohlander, S. K.; Espinosa, R.; Le Beau, M. M.; Rowley, J. D.; Diaz, M. O. (1992) A method for the rapid sequence-independent amplification of microdissected chromosomal material. Genomics 13, 1322–1324. Du Manoir, S.; Schröck, E.; Bentz, M.; Speicher, M. R.; Joos, S.; Ried, T.; Lichter, P.; Cremer, T. (1995) Quantitative analysis of comparative genomic hybridization. Cytometry 19, 27–41. Du Manoir, S.; Speicher, M. R.; Joos, S.; Schröck, E.; Popp, S.; Döhner, H.; Kovacs, G.; Robert-Nicoud, M.; Lichter, P.; Cremer, T. (1993) Detection of complete and partial chromosome gains and losses by comparative genomic in situ hybridization. Hum. Genet. 90, 590–610. Joos, S.; Scherthan, H.; Speicher, M. R.; Schlegel, J.; Cremer, T.; Lichter, P. (1993) Detection of amplified genomic sequences by reverse chromosome painting using genomic tumor DNA as probe. Hum. Genet. 90, 584–589.

5

Lichter, P.; Bentz, M.; du Manoir, S.; Joos, S. (1994) Comparative genomic hybridization. In: Verma, R; Babu S. (eds) Human Chromosomes. New York: McGraw Hill, 191–210. Lichter, P.; Cremer, T. Chromosome analysis by non-isotopic in situ hybridization. In: Human Cytogenetics: Constitutional Analysis. IRL Press. Lichter, P.; Tang, C. C.; Call, K.; Hermanson, G.; Evans, G. A.; Housman, D.; Ward, D. C. (1990) High resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones. Science 247, 64–69. Lundsteen, C.; Maahr, J.; Christensen, B.; Bryndorf, T.; Bentz, M.; Lichter, P.; Gerdes, T. (1995) Image analysis in comparative genomic hybridization. Cytometry 19, 42–50. Maniatis, T.; Fritsch, E. F.; Sambrook, J. (1986) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.

Kallioniemi, A.; Kallioniemi, O.-P.; Sudar, D.; Rutovitz, D.; Gray, J. W.; Waldman, F.; Pinkel, D. (1992) Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science 258, 818–821.

Piper, J.; Rutovitz, D.; Sudar, D.; Kallioniemi, A.; Kallioniemi, O.-P.; Waldmann, F. M.; Gray, J. W.; Pinkel, D. (1995) Computer image analysis of comparative genomic hybridization. Cytometry 19, 10–26.

Kallioniemi, O-P.; Kallioniemi, A; Piper, J.; Isola, J.; Waldman, F.; Gray, J. W.; Pinkel, D. (1994) Optimizing comparative genomic hybridization for analysis of DNA sequence copy number changes in solid tumors. Genes, Chromosomes and Cancer 10, 231–243.

Speicher, M. R.; du Manoir, S.; Schröck, E.; HoltgreveGrez, H.; Schoell, B.; Lengauer, C.; Cremer, T.; Ried, T. (1993) Molecular cytogenetic analysis of formalin-fixed, paraffin-embedded solid tumors by comparative genomic hybridization after universal DNA amplification. Hum. Mol. Genet. 2, 1907–1914.

Kallioniemi, O.-P.; Kallioniemi, A.; Sudar, D.; Rutovitz, D.; Gray, J. W.; Waldman, F.; Pinkel, D. (1993) Comparative genomic hybridization: a rapid new method for detecting and mapping DNA amplification in tumors. Seminars in Cancer Biology 4, 41–46.

Telenius, H.; Pelmear, A. H.; Tunnacliffe, A.; Carter, N. P.; Behmel, A.; Ferguson-Smith, M. A.; Nordenskjöld, M.; Pfragner, R. (1992) Ponder BAJP: Cytogenetic analysis by chromosome painting using DOP-PCR amplified flow-sorted chromosomes. Genes, Chromosomes and Cancer 4, 257–263.

Kawasaki, E. S. (1990) Sample preparation from blood, cells, and other fluids. In: Innis, M. A.; Gelfand, D. H.; Sninsky, J. J.; White, T. J. (eds) PCR Protocols: AGuide to Methods and Applications. San Diego, CA: Academic Press, 146–152.

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Identificationof chromosomesinmetaphasespreads and interphase nuclei with PRINS oligonucleotide primersandDIG- or rhodamine-labeled nucleotides D r. R . Seibl, R esearch L aboratories, Boehringer M annheim G m bH , Penz berg, G erm any.

PRimed I N Situ Labeling (PRIN S) is a fast alternative to traditional in situ hybridization (Koch et al., 1989). PRIN S starts with in situ hybridization (annealing) of an unlabeled synthetic oligonucleotide (e.g., one of the PRIN S oligonucleotide primers in Table 1) to denatured chromosomes in metaphase spreads or interphase nuclei on microscope slides. Then, the hybridized oligonucleotide serves as primer which a thermostable D N A polymerase (G osden et al., 1991) elongates in situ. D uring the primer elongation reaction, the polymerase incorporates nonradioactively labeled nucleotides into the newly synthesized D N A (G osden and H anratty, 1993). D epending on the label, the newly synthesized D N A may be detected by one of two methods: Indirect detection using the D IG PRIN S Reaction Set or direct detection using the Rhodamine PRIN S Reaction Set. ! I ndirect detection uses a fluorochrome-

conjugated anti-D IG antibody (for example, Anti-D igoxigenin-Fluorescein from the D IG PRIN S Reaction Set) to locate digoxigenin-(D IG -)labeled D N A. For indirect detection, we offer the D IG PRIN S Reaction Set, which combines the high sensitivity of digoxigenin labeling with the power of the PRIN S reaction. In the D igoxigenin PRIN S Reaction Set the sensitivity of the PRIN S reaction can be optimized by adjusting the dTTP-concentration individually according to experimental requirements. ! D irect detection

uses fluorochromelabeled nucleotides (e.g. RhodaminedU TP from the Rhodamine-PRIN S Reaction Set) that are directly visible under a fluorescence microscope. For direct detection, we offer the Rhodamine PRIN S Reaction Set, which combines the labeling intensity of our improved rhodamine which is the

optimal fluorochrome for the PRIN S reaction with the convenience of the PRIN S reaction. This combination produces extremely rapid results as there is no conjugate incubation step required and the best sensitivity in direct detection comparable to indirect detection. In the Rhodamine PRIN S Reaction Set, dTTP is provided at an optimized concentration in the Rhodamine Labeling Mix because dTTP variation has only minor influence on rhodamine labeling intensity.

Table 1: Specificity of PRINS oligonucleotide primers.

PRIN S oligonucleotide primer

Specific for

1

Satellite II D N A in the pericentromeric heterochromatin on the long arm of human chromosome 1

3

C entromere of human chromosome 3, Alpha satellite

7

C entromere of human chromosome 7, Alpha satellite

8

C entromere of human chromosome 8, Alpha satellite

9 (Sat)

Satellite III D N A in the pericentric heterochromatin on the proximal long arm of human chromosome 9

10

C entromere of human chromosome 10, Alpha satellite

11

C entromere of human chromosome 11, Alpha satellite

12

C entromere of human chromosome 12, Alpha satellite

14 + 22

C entromeres of human chromosomes 14 and 22, Alpha satellite

1 + 16

Satellite II D N A of human chromosomes 1 and 16 in both cases located to the proximal long arm of that chromosome.

17

C entromere of human chromosome 17, Alpha satellite

18

C entromere of human chromosome 18, Alpha satellite

X

C entromere of human chromosome X, Alpha satellite

Y

Long arm of human chromosome Y. Stains most of the long arm of the Y-chromosome.

T

Telomeres of all human chromosomes1

Control primer

All human chromosomes

5

1 Please note the Telomere Primer is only for use with the Digoxigenin PRINS Reaction Set, because the Rhodamine PRINS Reaction Set contains dTTP in the Rhodamine Labeling Mix.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

C ompared to traditional ISH methods (where probe labeling is performed before the probe is added to the slide), PRIN S has two advantages: ! C onv enience. PRIN S circumvents the

laborious probe isolation and labeling needed in other approaches. ! Speed. Since high probe concentrations

are possible, hybridization and chain elongation (in one reaction) usually takes 30 min. For many PRIN S primer the reaction time can even be reduced. The complete procedure, including fluorescent antibody detection, is complete in approximately 2 h. When using the Rhodamine PRIN S Reaction Set for direct detection results are already available after 60 min. This article describes the use of the PRIN S Reaction Set, the Rhodamine-PRIN S Reaction Set, and PRIN S oligonucleotide primers to rapidly identify human chromosomes. It also details how to combine PRIN S and FISH (fluorescence in situ hybridization) for a multiple labeling experiment.

5

80

I. Preparation of slides " 1 U sing standard methods (e.g. G osden,

1994; Roonea and C zepulkowski, 1992), prepare fresh microscope slides containing fixed metaphase chromosomes and/ or interphase nuclei. C aution: For best results, prepare the slides containing the fixed m etaphase chrom osom es either im m ediately before use or on the day before the PR I N S reaction. I f slides hav e been prepared a day early, store them at 4° C until use. Such slides m ay be used in the PR I N S reaction w ithout form am ide pretreatm ent (Step 2 below ). 2 (O ptional) If slides have been stored for "

3 or more days at room temperature in 70% ethanol at 4°C , denature the preparations with formamide directly before the PRIN S reaction. For the formamide treatment, perform the following steps: ! Prepare a solution containing: – 70 ml deionized formamide – 10 ml 20 x SSC – 10 ml 500 mM sodium phosphate buffer, pH 7.0 – 10 ml H 2O ! Adjust the pH of the formamide solution to 7.0 with H C l. ! Incubate the slides for 45 s at 68° C in the formamide solution. Check the temperature of the formamide solution, as it is important that it be 68°C. ! Wash the slides in a series of ice-cold ethanol solutions (70% , then 90% , then 100% ethanol). C aution: T he ethanol solutions m ust be ice-cold during this step.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

II. Preparation of PRINS reaction mixes N ote: For a single reaction under a 22 x 22 m m cov erslip, prepare 30 µl of D I G PR I N S reaction m ix or R hodam ine-PR I N S reaction m ix. A djust the v olum e of the reaction m ix (but not the concentration of the com ponents) if you are preparing m ore than one reaction, or if you use cov erslips of a different siz e. For 18 x 18 m m cov erslips, prepare 15 m l reaction m ix per reaction; for 24 x 60 m m or 22 x 40 m m cov erslips, prepare 60 m l per reaction. " 1 D epending on the type of PRIN S

reaction desired, do one of the following: A. I f you want to use D igoxigenin for in situ labeling and detect the PRIN S target by indirect fluorescence (immunological) methods, prepare 30 µl of D IG -PRIN S reaction mix (for each reaction) by adding the following components to a sterile microcentrifuge tube (on ice): N ote: M ost of the com ponents (num bered v ials) used in the D I G -PR I N S reaction m ix are from the PR I N S R eaction Set. ! 13.5 µl sterile redistilled H 2O . ! 3.0 µl 10 x concentrated PRIN S

reaction buffer (PRIN S vial 3).

! 3.0 µl 10 x concentrated D IG -PRIN S

labeling mix (500 µM each of dATP, dC TP, and dG TP; 50 µM D IG dU TP; 50% glycerol) (PRIN S vial 1). ! 5.0 µl PRIN S oligonucleotide primer [either control primer (PRIN S vial 4) or a human chromosome-specific PRIN S oligonucleotide primer (Table 1). ! 3.0 µl 450 mM dTTP (PRIN S vial 2). C aution: T he concentration of dT T P used in the D I G -PR I N S reaction m ix depends upon the PR I N S oligonucleotide prim er used. U sing the PR I N S O ligonucleotide Prim er T, no -dT T P has to be added to the reaction m ix. For the PRIN S O ligonucleotide Primer 9, the final concentration of dTTP in the DIG-PRIN S reaction mix must be 90 µM. For all other PR I N S prim ers listed in Table 1 the final concentration of dT T P in the D I G -PR I N S reaction m ix m ust be 45 µM . T he correct dT T P concentration for other prim ers m ust be determ ined em pirically.

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! T he dT T P concentration can be

adjusted indiv idually to increase the signal strength by reducing the dT T P concentration or to decrease the signal strength by increasing the dT T P concentration. ! 2.5 µl Taq DN A Polymerase (1 unit/µl).

B. I f you want to use rhodamine-labeled D N A and detect the PRIN S target by direct fluorescence methods, prepare 30 µl of Rhodamine-PRIN S reaction mix (for each reaction) by adding the following components to a sterile microcentrifuge tube (on ice): N ote: M ost of the com ponents (num bered v ials) used in the R hodam inePR I N S reaction m ix are from the R hodam ine-PR I N S R eaction Set. ! 16.5 µl sterile redistilled H 2O . ! 3.0 µl 10 x concentrated PRIN S

reaction buffer (Rh-PRIN S vial 2).

! 3.0 µl 10 x concentrated Rhodamine-

PRIN S labeling mix (500 µM each of dATP, dC TP, and dG TP; 50 µM dTTP; 10 µM rhodamine-dU TP; 50% glycerol) (Rh-PRIN S vial 1). Note: The correct dTTP concentration (5 µM) for most primers is provided as part of the Rhodamine-PRIN S labeling mix (in the RhodaminePRIN S Reaction Set). Variations in dTTP do not significantly affect the intensity of rhodamine labeling. ! Since dTTP is included in the Rhodamine-PRIN S labeling mix and because for the PRIN S O ligonucleotide Primer T, no dTTP has to be added to the reaction mix, the T primer is incompatible with the components of the Rhodamine-PRIN S Reaction Set.

5

! 5.0 µl PRIN S oligonucleotide primer

[either control primer (Rh-PRIN S vial 3) or a human chromosomespecific PRIN S oligonucleotide primer (Table 1). ! 2.5 µl Taq DN A Polymerase (1 unit/µl). 2 G ently vortex the reaction solution to "

mix the components.

" 3 C entrifuge the reaction mixture briefly

to collect the liquid on the bottom of the tube.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

III. Primer annealing and elongation " 1 Place the dry slides containing fixed

! I f slides are 3 or more days old and

chromosomes and/ or interphase nuclei on either a heating block or a special thermal cycler for slides. N ote: To guarantee specificity and sensitiv ity of the reaction, the denaturation step and the annealing/ elongation step need precisely controlled elev ated tem peratures. For reproducible results, w e recom m end that, instead of a heating block , you use a tem perature cycler that is specially designed for slides, such as the O m niSlide or O m niG ene Tem perature C ycler (H ybaid L td., Teddington, M iddlesex, England) or an equiv alent instrum ent from another m anufacturer.

have been treated with formamide, skip this step, leave the heating block set at 60°C and go to Step 5. 5 Reduce the temperature of the heating " block to 60° C and incubate the slides for 30 min at 60°C while the primer anneals and the polymerase elongates it. C aution: Be sure the tem perature of the slides is exactly as indicated for the indiv idual prim ers. Ev en a few seconds of exposure to a tem perature below the stringent annealing tem perature can lead to an unw anted back ground signal. T he slides are usually at the correct tem perature w hen the surface of the heating block reaches 60°C .

2 D epending on the age of the slides, do "

one of the following: ! I f slides are freshly prepared (one day old or less), incubate them at 94° C for 1 min. ! I f slides are 3 or more days old and have been treated with formamide (Procedure I, Step 2 above), incubate them at 60°C for 1 min. " 3 Without removing the slides from the heating block, do the following: ! O nto each sample, immediately pipette: – 25 µl of D IG -PRIN S reaction mix or – 27 µl of Rhodamine-PRIN S reaction mix ! C over sample with a 22 x 22 mm coverslip. C aution: I f you use cov erslips of a different siz e than 22 x 22 m m , adjust the v olum e of the D I G -PR I N S or R hodam ine-PR I N S reaction m ix in this step so it cov ers the sam ple w ell and com pletely w ets the cov erslip.

5

4 D epending on the age of the slides, do "

one of the following: ! I f slides are freshly prepared (1 day old or less), incubate them at 91°–94°C for 3 min to denature the chromosomal D N A, then go to Step 5. C aution: T he slides generally reach 91°–94° C w hen the surface of a heating block reaches 94°–97° C . H ow ev er, if you are using a tem perature cycler designed for slides, the cycler may autom atically tak e into account this tem perature differential.

82

Note: The stringent annealing/ elongation temperature for the PRIN S reaction depends on the sequence of the primer used. T he annealing tem perature in the PR I N S reaction using the O ligonucleotide Prim er T is 60°C . T he annealing tem perature for all other PR I N S O ligonucleotide Prim er in the PR I N S reaction is 60°–65°C . 6 Remove the slides from the heating "

block and place them immediately in a C oplin jar containing stop buffer which has been prewarmed to the temperature of the hybridization/ elongation reaction (60°C ). " 7 Stop the reaction by washing the slides for 3–5 min at the temperature of the hybridization/ elongation reaction (60° C ) with prewarmed stop buffer. N ote: C ov erslips should drop off the slides during this step. Caution: Be sure the temperature remains the temperature of the hybridization/ elongation reaction (60°C) throughout the wash.

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IV. Detection of hybridized sequences

V. Combining PRINS and FISH

" 1 In a C oplin jar, wash the slides as follows: ! 3 x 5 min at 37°C with wash buffer

" 1 Label a D N A probe with digoxigenin,

[0.2% Tween® 20 in phosphate buffered saline (PBS)]. ! About 1 min with PBS at room temperature. 2 D epending on the label used in the " PRIN Sreaction, do one of the following: ! I f you produced D IG -labeled D N A in Procedure III, go to Step 3. ! I f you produced rhodamine-labeled D N A in Procedure III, go to Step 6. " 3 Prepare the antibody working solution by diluting the Anti-D igoxigeninFluorescein conjugate (PRIN S vial 5) 1:1000 with PBS containing 1% blocking solution. Note: Anti-DIG-Fluorescein conjugate is a included in the PRIN S Reaction Set. 10 x blocking solution is included in the DIG Wash and Block Set*; it contains 100 mM maleic acid (pH 7.5), 150 mM N aCl, and 10% blocking reagent. PBS containing 1% blocking solution can be prepared by diluting the 10 x blocking solution 1:10 with PBS. 4 In a C oplin jar, incubate the slides with " antibody working solution for 30 min in the dark at 37° C . 5 Repeat Step 1 washes. " 6 For counterstaining, add the counter" stain working solution (e.g., PBS containing either 20 ng/ ml propidium iodide or 20 ng/ ml D API) to the C oplin jar and incubate the slides with the counterstain for 5 min in the dark at room temperature. C aution: I f the target D N A is labeled w ith rhodam ine-dU T P, do not use propidium iodide counterstain, since the em ission of propidium iodide and rhodam ine ov erlap. U se D A PI as a counterstain for rhodam ine-labeled D N A . ! Wash the slides for 2–3 min under running water. " 7 Air-dry the slides in the dark. 8 Prepare the slides for viewing by doing " the following: ! Apply 20 µl antifade solution [e.g., Vectashield (Vector) or Slow FadeLight (Molecular Probes)] to each slide. ! Add a coverslip. N ote: For long term storage of the slides seal the edges of the cov erslip w ith nail polish and store in the dark at –20° C . 9 Analyze the slides under a fluorescence " microscope with the appropriate filters.

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biotin, or any fluorochrome (except the one used in the PRIN S procedure), according to procedures described in C hapter 4 of this manual. 2 Perform the PRIN S reaction as described " in Procedure I–III of this article. " 3 After washing the PRIN S slides in stop buffer (Procedure III, Step 6), dehydrate the slides in an ethanol series (increasing concentrations of ice-cold ethanol). C aution: T he ethanol solutions m ust be ice-cold during this step. 4 H ybridize the labeled D N A probe "

(from Step 1) to the PRIN S slides, according to FISH procedures described elsewhere (Procedure IIA, IIB, or IIC from the article “In situ hybridization to human metaphase chromosomes using D IG -, biotin-, or fluorochrome-labeled D N A probes and detection with fluorochrome conjugates”, in C hapter 5 of this manual, pages 62, 63). Modify the hybridization procedure as follows: ! D enature the labeled probe D N A before adding it to the slides. ! D o not heat the slides to 80°C after adding the probe to the slides. 5 After the incubation step, wash the slides " with the formamide/SSC washes described in the FISH procedure, then perform a final wash in PBS. 6 D epending on the label used in the " PRIN S reaction, do one of the following: ! For PRIN S targets labeled with D IG dU TP, perform the PRIN S antibody detection and wash steps as above (this article, Procedure IV, Steps 3–5). ! For PRIN S targets labeled with rhodamine-dU TP, skip this step and go to Step 7. " 7 D epending on the label used in the FISH procedure, do one of the following: ! For biotin- or digoxigenin-labeled FISH probes, perform the immunological detection step according to procedures described elsewhere (Procedure IIIA1/ IIIA2 or IIIB1/ IIIB2 from the article “In situ hybridization to human metaphase chromosomes using D IG -, biotin-, or fluorochromelabeled D N A probes and detection with fluorochrome conjugates”, in C hapter 5 of this manual, page 64). ! For fluorochrome-labeled FISH probes, skip this step and go to Step 8.

5

*Sold under the tradename of Genius in the US.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

8 C ounterstain and prepare the slides as "

above (this article, Procedure IV, steps 6–7). " 9 Analyze the slides under a fluorescence microscope with the appropriate filters.

VI. Troubleshooting guide for PRINS U se the following tips to diagnose and correct commonly occurring problems in the PRIN S procedure described above. A. If signal is weak or missing, then:

Possible cause

Possible solution

D enaturation temperature does not reach Increase the temperature of the block 91°–94°C at the surface of the slide. slightly during denaturation. Annealing/ elongation temperature is too high.

Lower the annealing/ elongation temperature by 3°C and repeat experiment. If signal is inadequate, reduce the temperature further (in 3°C steps).

(For D IG -labeling only) N ot enough D IG -dU TP was incorporated into sample.

1. Reduce the dTTP concentration in the reaction solution to 30 µM, 15 µM or zero by adding 2 µl, 1 µl, or no dTTP solution (D IG PRIN S Reaction Set vial 2) to each 30 µl reaction

Primer concentration too low

1. Increase the amount of primer in the reaction solution. U se the control primer (D IG PRIN S Reaction Set vial 4) to check the sensitivity of the reaction.

B. If signal is strong, then:

5

84

Possible cause

Possible solution

(For D IG -labeling only) Too much D IG -dU TP was incorporated into sample.

1. Increase the dTTP concentration in the reaction solution to 60 µM, 90 µM or 135 µM by adding 4 µl, 6 µl, or 9 µl dTTP solution (D IG PRIN S Reaction Set vial 2) to each 30 µl reaction

Primer concentration too high

1. D ecrease the amount of primer in the reaction solution. U se the control primer (D IG PRIN S Reaction Set vial 4) to check the sensitivity of the reaction.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

C. If chromosomes or nuclei contain unwanted background signals, then:

Possible cause

Possible solution

The slide was exposed to temperatures below the stringent annealing/ elongation temperature for a few seconds after the denaturation step.

D o not allow the temperature on the slide to ever fall below the stringent annealing/ elongation temperature.

Annealing/ elongation temperature is too low.

Raise the annealing/ elongation temperature by 3°C and repeat experiment. If background is still high, increase the temperature further (in 3°C steps) until the reaction is specific.

The slide was exposed to temperatures below the stringent annealing/ elongation temperature for a few seconds between the elongation step and the addition of stop buffer.

Prewarm the stop buffer to the stringent annealing/ elongation temperature and transfer the slides directly from the heating block to the warm stop solution.

Primer-independent elongation of nicks and chromosomal breaks occurred. Such nicks and breaks can occur during long term storage of the chromosomal preparation.

Perform a control PRIN S reaction without added primer. If you obtain a signal, either block the 3' ends of the chromosomal breaks by incubating the chromosomal preparations with D N A polymerase and a mixture of ddN TPS or (preferably) discard the chromosomal preparations and prepare fresh chromosomes or fresh chromosomal spreads.

(For D IG -labeling only) When the primary signal is strong, minor primerdependent signals may be seen at secondary binding sites for the primer.

Increase the dTTP concentration in the reaction solution to 60 µM, 90 µM or 135 µM by adding 4 µl, 6 µl, or 9 µl dTTP solution (D IG PRIN S Reaction Set vial 2) to each 30 µl reaction or D ecrease the amount of primer in the reaction solution.

5

D. If the entire slide (not just the chromosomes and nuclei) contain unwanted background signals, then:

Possible cause

Possible solution

(For D IG -labeling only) The antibody conjugate (anti-digoxigenin-fluorescein) bound nonspecifically to the slide or to cellular proteins and matrix in the chromosomal preparation.

Perform a control detection reaction by incubating an unhybridized sample slide directly with the antibody conjugate, then washing and analyzing the slide as in Procedure IV. I f background is observed, either increase the number and duration of the washing steps or (preferably) repeat the chromosomal preparation to remove excess cellular proteins and matrix responsible for nonspecific binding.

D IG -dU TP or rhodamine-dU TP bound nonspecifically during the PRIN S reaction.

Perform a control PRIN S reaction in the absence of Taq polymerase and primer. I f background is observed, either increase the number and duration of the washes or (preferably) repeat the chromosomal preparation to remove excess cellular proteins and matrix responsible for nonspecific binding.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

Results

a

b

#

Figure 1: PRINS labeling and indirect fluorescent detection of human chromosomes. The PRINS primers specific for (Panel a) human chromosome 11 and (Panel b) human chromosome 7 were hybridized to human chromosomal preparations. The hybridized primers were elongated in situ and the newly synthesized DNA labeled with DIG-dUTP according to the procedure described in the text. Labeled DNA was visualized with anti-DIG-fluorescein. Propidium iodide was used as a counterstain.

a

5

b1

#

$ Figure 2: Rhodamine PRINS labeling and direct

fluorescent detection of human chromosomes. The PRINS primers specific for (Panel a) human chromosome 1 and (Panel b1, b2) human chromosome 9 were hybridized to human chromosomal preparations. The hybridized primers were used to label target DNA in situ with rhodamine-dUTP according to the procedure described in the text. Labeled DNA was viewed directly under a fluorescence microscope. DAPI was used as a counterstain.

b2

References Gosden, J. R., ed. (1994) Chromosome Analysis Protocols (Methods Mol. Biol., Vol. 29). Totowa, NJ: Humana Press. Gosden, J.; Hanratty, D.; Starling, J.; Fantes, J.; Mitchell, A.; Porteus, D. (1991) Oligonucleotide primed in situ DNA synthesis (PRINS): A method for chromosome mapping, banding and investigation of sequence organization. Cytogenet. Cell Genet. 57, 100–104.

86

Gosden, J.; Hanratty, D. (1993) PCR in situ: A rapid alternative to in situ hybridization for mapping short, low-copy number sequences without isotopes. BioTechniques 15, 78–80. Koch, J. E.; Kølvraa, S.; Petersen, K. B.; Gregersen, N.; Bolund, L. (1989) Oligonucleotide priming methods for the chromosome-specific labeling of alpha satellite DNA in situ. Chromosoma (Berl.) 98, 259–265. Roonea, D. E.; Czepulkowski, B. H., eds. (1992) Human Cytogenetics, A Practical Approach, Vol. 1. Oxford, England: IRL Press.

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Reagents available from Boehringer Mannheim for PRINS Product

Description

Cat. No.

DIGPRINS Reaction Set

For 40 PRINS labeling reactions and 5 control reactions

1 695 932

The D IG PRIN S Reaction Set includes a detailed working procedure and contains: Reagent

Description

Kit component

10 x Concentrated PRINS labeling mix

dNTPs including digoxigenin-11-dUTP

One vial No. 1, 135 µl

dTTP solution

450 µM dTTP

One vial No. 2, 200 µl

10 x Concentrated PRINS reaction buffer

Buffer for 45 reactions

One vial No. 3, 150 µl

PRINS Control Oligonucleotide Primer

Primer for 5 control reactions

Anti-Digoxigenin-Fluorescein Conjugate, Fab fragments

Antibody (200 µg/ml) for 22 detection reactions in a Coplin jar

Product

Description

Cat. No.

Rhodamine PRINS Reaction Set

For 40 PRINS labeling reactions and 5 control reactions

1 768 514

One vial No. 4 Two vials No. 5, 2 x 550 µl

The Rhodamine PRIN S Reaction Set includes a detailed working procedure and contains: Reagent

Description

Kit component

10 x Concentrated Rhodamine PRINS labeling mix

dNTPs including Rhodamine-dUTP

One vial No. 1, 135 µl

10 x Concentrated PRINS reaction buffer

Buffer for 50 reactions

One vial No. 2, 150 µl

PRINS Control Oligonucleotide Primer

Primer for 5 control reactions

One vial No. 3, 25 µl

5

PRIN S oligonucleotide primers The following PRIN S oligonucleotide primers are available* (Pack size 100 µl each): Primer

Cat. No.

PRINS oligonucleotide primer 1, specific for human chromosome 1

1 695 959

PRINS oligonucleotide primer 1+16, specific for human chromosome 1+16

1768 549

PRINS oligonucleotide primer 3, specific for chromosome 3

1 695 967

PRINS oligonucleotide primer 7, specific for human chromosome 7

1 695 975

PRINS oligonucleotide primer 8, specific for human chromosome 8

1 695 983

PRINS oligonucleotide primer 9 (Sat), specific for human chromosome 9

1768 565

PRINS oligonucleotide primer 10, specific for human chromosome 10

1768 522

PRINS oligonucleotide primer 11, specific for human chromosome 11

1 695 991

PRINS oligonucleotide primer 12, specific for human chromosome 12

1 696 009

PRINS oligonucleotide primer 14+22, specific for human chromosome 14+22

1768 557

PRINS oligonucleotide primer 17, specific for human chromosome 17

1 696 017

PRINS oligonucleotide primer 18, specific for human chromosome 18

1 696 025

PRINS oligonucleotide primer X, specific for the human X chromosome

1 696 033

PRINS oligonucleotide primer Y, specific for the human Y chromosome

1 696 041

PRINS oligonucleotide primer T, specific for human telomeres

1 699 199

PRINS control oligonucleotide primer, specific for all human chromosomes

1 699 172

CONTENTS

INDEX

* Please note the Telomer Primer is only for use with the Digoxigenin PRINS Reaction Set because the Rhodamine PRINS Reaction Set contains dTTP in the Labeling Mix.

87

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

Identification of chromosomes in metaphase spreads with DIG- or fluorescein-labeled human chromosome-specific satellite DNA probes D r. G . Sagner, R esearch L aboratories, Boehringer M annheim G m bH , Penz berg, G erm any.

Fluorescence in situ hybridization (FISH ) with human chromosome-specific satellite D N A probes is a sensitive technique for identifying individual human chromosomes. Applications include: " Analysis of aneuploidies in normal or tumor cells " Identification of chromosomes in hybrid cells " Production of genetic linkage maps Especially useful for such applications are fluorescein- or digoxigenin-labeled probes which recognize alphoid sequence variants that occur at the centromere of specific chromosomes (Willard and Waye, 1987; C hoo, K. H . et al., 1991). Fluoresceinlabeled probes allow rapid, direct detection of a single chromosome. D IG -labeled probes allow sensitive, indirect detection of a chromosome with a variety of fluorochrome-labeled antibodies. Both fluorescein- and D IG -labeled probes can be combined in a multicolor labeling of different chromosomes on a single preparation.

5

This article describes the use of such prelabeled probes to identify human chromosomes in chromosomal spreads from whole blood or from cell culture.

88

I. Preparation of metaphase chromosomes ! 1 Prepare chromosomes from either whole

blood or cell culture as follows:

" For w hole blood, do these steps: " Mix 5 ml whole blood with 5 ml PBS. " Pipette 7.5 ml of lymphocyte separa-

tion medium into a 50 ml centrifuge tube. " C arefully layer blood-PBS mixture atop separation medium. " C entrifuge for 30 min at 800 x g. " Remove the interphase (middle part of the centrifuged mixture) that contains the lymphocytes. " Place the lymphocytes in a fresh centrifuge tube. " Mix lymphocytes with 50 ml PBS. " C entrifuge for 10 min at 420 x g. " D iscard the supernatant and resuspend cells at approximately 106 cells per ml in chromosome medium (G ibco). N ote: T his step should require about 5 m l chrom osom e m edium . " G o to Step 2. " For cells in culture, do these steps: " H arvest cells by centrifugation. " Resuspend cells at approximately 106 cells per ml in chromosome medium. " G o to Step 2. 2 Incubate cells in chromosome medium ! for 71 h in a C O 2 incubator at 37°C and 7% C O 2. ! 3 Add 12 µl C olcemid ® per ml medium. 4 Incubate cells for 1 h at 37°C and 7% ! C O 2. 5 C entrifuge cells (210 x g) for 10 min at ! 4° C . 6 With a pipette, remove supernatant until ! only 1 ml remains atop the cells.

CONTENTS

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

! 7 Resuspend the pellet in the remaining

supernatant.

8 Slowly add to the resuspended cells 10 ml !

of prewarmed (37° C ) 75 mM KC l.

! 9 G ently mix and incubate for 12–20 min

at 37° C .

10 To the mixture, add 7 drops of chilled !

methanol-acetic acid fixative (3 parts methanol to 1 part acetic acid, precooled to –20° C ). G ently mix. 11 C entrifuge mixture (210 x g) for 10 min ! at 4° C . ! 12 With a pipette, remove supernatant until only 1 ml remains atop the pellet. 13 C arefully resuspend the pellet in the ! remaining supernatant and add 1 ml precooled (–20°C ) methanol-acetic acid fixative. G ently mix. ! 14 Add an additional 5–10 ml precooled (–20° C ) methanol-acetic acid fixative. Mix thoroughly. ! 15 Fix chromosomes for at least 30 min at –20° C . 16 Repeat Steps 11–15 three to five times. ! ! 17 Store the metaphase chromosomes at least 24 h in methanol-acetic acid fixative at –20°C . 18 D o either of the following: ! " G o to Procedure II. " Store metaphase chromosomes in methanol-acetic acid fixative at –20°C up to several months.

II. Preparation of chromosomal spreads ! 1 Pretreat slides as follows: " Wash slides briefly in a 1:1 mixture of

100% ethanol and ether.

" Air dry slides. " Wash briefly in redistilled H 2O . (D o

not dry.)

2 Centrifuge metaphase chromosomes from !

Procedure I (210 x g) for 10 min at 4°C.

! 3 Remove and discard all supernatant. 4 Resuspend chromosomal pellet in 1–2 ml !

of precooled (–20°C ) methanol-acetic acid fixative. 5 D rop the metaphase chromosomes onto ! a moist, pretreated slide from a height of approximately 50 cm. 6 Allow slides to air dry. ! ! 7 For optimal metaphase spreads, store slides at least 5 days in 70% ethanol at 4° C . N ote: You m ay store slides up to sev eral w eek s in 70% ethanol at 4° C .

CONTENTS

INDEX

III. In situ hybridization with chromosome-specific DNA probes N ote: Perform the follow ing procedure in a 50 m l C oplin jar w hich can hold a m axim um of 8 slides. C aution: Prew arm all solutions in the follow ing procedure to the appropriate tem perature before using them . ! 1 Remove slides from the 70% ethanol

storage solution (from Procedure II, Step 7). 2 Air dry slides completely (approxi! mately 30 min). ! 3 For each slide, prepare 20 ml hybridization solution containing: " 10–13 µl deionized formamide (final concentration: 50–65% formamide) Note: Different probes require different concentrations of deionized formamide. See Table 1 for the correct formamide concentration to use with each probe. " 5 µl 4 x concentrated hybridization buffer [8 x SSC ; 40% (v/ v) dextran sulfate; 4 mg/ ml MB-grade D N A (from fish sperm); pH 7.0]. " 1–2 µl (20–40 ng) human chromosome-specific satellite D N A probe, either fluorescein- or D IG -labeled. " Enough redistilled H 2O to make a total volume of 20 µl. C aution: Tw enty µl is enough hybridiz ation solution if you are using a 24 x 24 m m cov erslip in Step 7 below . I f you use a different siz e cov erslip, adjust the v olum e of the hybridiz ation solution (but not the concentration of the com ponents) accordingly. 4 Preheat a glass tray big enough to hold ! all slides in a 72°C oven. 5 Prewarm a moist chamber to 37°C . ! 6 Pipette 20 µl hybridization solution ! onto each slide, add a 24 x 24 mm coverslip, and seal the coverslip to the slide with rubber cement. ! 7 Place the prepared slides onto the prewarmed glass tray in the oven and incubate 5–10 min at 72°C to denature D N A. 8 Transfer slides to the prewarmed moist ! chamber and incubate overnight at 37°C .

5

89

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

IV. Detection of hybridized sequences IVA. For detection of fluorescein-labeled chromosome-specific probes

IVB. For detection of DIG-labeled chromosome-specific probes

N ote: Perform Steps 1 and 2 in a C oplin jar.

! 1 After hybridization, wash slides in a

! 1 After hybridization, wash slides ac-

C oplin jar according to one of the following wash schemes: " 15 min at 40°–48°C with 1 x SSC containing 50% formamide, pH 7.0; then: 2 x 5 min at room temperature with 2 x SSC , pH 7.0 or " 15 min at 37°C with 2 x SSC , pH 7.0. N ote: D ifferent probes require different w ashes. See Table 1 for the correct w ashes to use w ith each probe. 2 (O ptional) Incubate slides for 30 min at ! 37° C with 50 ml PBS containing 1% blocking reagent. ! 3 Just before use, prepare 100 µl of a 1 µg/ ml working solution of anti-D IG -fluorophore conjugate in PBS containing 1% blocking reagent. N ote: Each slide to be analyz ed requires 100 µl of antibody w ork ing solution. N ote: A nti-D I G -fluorophore conjugate can be Anti-DIG-Fluorescein, Anti-DIGA M C A , or A nti-D I G -R hodam ine. 4 Add 100 µl freshly prepared antibody ! working solution to each slide and cover the complete slide with a coverslip. 5 Incubate slides for 30 min at 37°C in a ! moist chamber. 6 In a C oplin jar, wash slides for 3 x 5 min ! at room temperature with 2 x SSC containing 0.1% Tween ® 20. ! 7 C ounterstain, mount and view slides as for fluorescein-labeled probes (Steps 2–7, Procedure IVA). Exception: For experim ents using antiD I G -rhodam ine conjugates, use D A PI rather than propidium iodide as a counterstain. T he em ission w av elength of propidium iodide is too near that of rhodam ine. N ote: M axim um em ission w av elength for A M C A is 474 nm ; for rhodam ine, 570 nm .

cording to one of the following wash schemes: " 15 min at 30°–48°C with 1 x SSC containing 50% formamide, pH 7.0; then: 2 x 5 min at room temperature with 2 x SSC , pH 7.0 or " 15 min at 37°C with 2 x SSC , pH 7.0. N ote: D ifferent probes require different w ashes. See Table 1 for the correct w ashes to use w ith each probe. 2 For counterstaining, add 50 ml of either ! propidium iodide solution (100 ng/ ml propidium iodide in PBS) or D API solution (100 ng/ ml D API in PBS) to the slides, then incubate the slides for 2–5 min in the dark at room temperature. ! 3 Wash slides 2–3 min under running water. 4 Air dry the slides in the dark. ! 5 Apply 20 µl antifade solution [e.g., 2% ! D ABC O (w/ v) in PBS containing 50% glycerol] to each slide. 6 Add a 24 x 24 mm coverslip. ! N ote: For long term storage of the slides, seal the edges of the cov erslip w ith nail polish and store in the dark at –20° C . ! 7 Analyze the slides under a fluorescence microscope with the appropriate filters. N ote: M axim um em ission w av elength for fluorescein (FL U O S) is 523 nm ; for propidium iodide, 610 nm ; and for D A PI , 470 nm .

5

90

CONTENTS

INDEX

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

Probe1 for chromosomes

Labeled with

1

D IG 2 FLU O S

65% 65%

43°C ; 1x SSC + 50% formamide 43°C ; 1x SSC + 50% formamide

RT; 2 x SSC RT; 2 x SSC

2

D IG FLU O S

65% 65%

48°C ; 1x SSC + 50% formamide 48°C ; 1x SSC + 50% formamide

RT; 2 x SSC RT; 2 x SSC

3

D IG

65%

43°C ; 1x SSC + 50% formamide

RT; 2 x SSC

4

D IG

65%

43°C ; 1x SSC + 50% formamide

RT; 2 x SSC

5 + 1 + 19

D IG FLU O S

65% 65%

46°C ; 1x SSC + 50% formamide 43°C ; 1x SSC + 50% formamide

RT; 2 x SSC RT; 2 x SSC

6

D IG

65%

43°C ; 1x SSC + 50% formamide

RT; 2 x SSC

7

D IG FLU O S

65% 65%

46°C ; 1x SSC + 50% formamide 43°C ; 1x SSC + 50% formamide

RT; 2 x SSC RT; 2 x SSC

8

D IG FLU O S

65% 65%

43°C ; 1x SSC + 50% formamide 43°C ; 1x SSC + 50% formamide

RT; 2 x SSC RT; 2 x SSC

9

D IG

65%

46°C ; 1x SSC + 50% formamide

RT; 2 x SSC

10

D IG FLU O S

65% 65%

46°C ; 1x SSC + 50% formamide 43°C ; 1x SSC + 50% formamide

RT; 2 x SSC RT; 2 x SSC

11

D IG FLU O S

65% 65%

43°C ; 1x SSC + 50% formamide 43°C ; 1x SSC + 50% formamide

RT; 2 x SSC RT; 2 x SSC

12

D IG FLU O S

65% 65%

46°C ; 1x SSC + 50% formamide 43°C ; 1x SSC + 50% formamide

RT; 2 x SSC RT; 2 x SSC

13 + 21

D IG

65%

43°C ; 1x SSC + 50% formamide

RT; 2 x SSC

14 + 22

D IG FLU O S

65% 65%

43°C ; 1x SSC + 50% formamide 43°C ; 1x SSC + 50% formamide

RT; 2 x SSC RT; 2 x SSC

15

D IG

65%

43°C ; 1x SSC + 50% formamide

RT; 2 x SSC

16

D IG

65%

48°C ; 1x SSC + 50% formamide

RT; 2 x SSC

17

D IG FLU O S

65% 65%

48°C ; 1x SSC + 50% formamide 30°C ; 1x SSC + 50% formamide

RT; 2 x SSC RT; 2 x SSC

18

D IG FLU O S

65% 65%

46°C ; 1x SSC + 50% formamide 46°C ; 1x SSC + 50% formamide

RT; 2 x SSC RT; 2 x SSC

20

D IG FLU O S

65% 65%

48°C ; 1x SSC + 50% formamide 43°C ; 1x SSC + 50% formamide

RT; 2 x SSC RT; 2 x SSC

22

D IG

65%

40°C ; 1x SSC + 50% formamide

RT; 2 x SSC

X

D IG FLU O S

65% 65%

46°C ; 1x SSC + 50% formamide 46°C ; 1x SSC + 50% formamide

RT; 2 x SSC RT; 2 x SSC

Y

D IG FLU O S

50% 50%

37°C ; 2 x SSC 37° C ; 2 x SSC

O mit O mit

All human

D IG FLU O S

50% 50%

37°C ; 2 x SSC 37° C ; 2 x SSC

O mit O mit

Formamide (%) in hybridization solution

Posthybridization wash conditions Wash 1 (15 min) Wash 2 (2 x 5 min)

1 The probes all recognize a pattern of alphoid DNA on specific chromosomes, except for the chromosome 1 probe (which recognizes satellite III DNA on chromosome 1) and the chromosome Y probe (which recognizes a repeat on the long arm of chromosome Y). 2 Abbreviations used: DIG, digoxigenin; FLUOS, 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester; 1xSSC, buffer (pH 7.0) containing 150 mM NaCl and 15 mM sodium citrate; RT, room temperature.

CONTENTS

INDEX

5

Table 1: Hybridization parameters for human chromosome-specific satellite DNA probes. Use the data given in this table in Procedures III and IV of this article. All probes are available from Boehringer Mannheim.

91

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

Results

a

b

$ # Figure 1: FISH of human chromosome-specific

satellite DNA probes to metaphase spreads. Fluorescence signals have been pseudocolored with a CCD camera and a digital imaging system. In Panel a, a DIG-labeled probe specific for chromosomes 5 + 1 + 19 was detected with antiDIG-rhodamine. Chromosomes were counterstained with DAPI. In Panel b, a DIG-labeled probe specific for chromosome 12 was detected with anti-DIGfluorescein. Chromosomes were counterstained with propidium iodide. Panel c shows the direct detection of chromosome 12 sequences with a fluorescein-labeled probe. Chromosomes were counterstained with propidium iodide.

5

c

References Waye, J. S.; Willard, H. F. (1987) Nucleotide sequence heterogeneity of alpha satellite repetitive DNA: a survey of alphoid sequences from different human chromosomes. Nucl. Acids Res. 15, 7549–7569. Choo, K. H.; Vissel, B.; Nagy, A.; Earle, E.; Kalitsis, P. (1991) A survey of the genomic distribution of alpha satellite DNA on all the human chromosomes, and derivation of a new consensus sequence. Nucl. Acids Res. 19, 1179–1182.

92

CONTENTS

INDEX

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

Reagents available from Boehringer Mannheim for this procedure Reagent

Description

Cat. No.

Pack size

DNA, MB-grade from fish sperm

It prevents unspecific hybridization in all types of membrane or in situ hybridization

1 467 140

500 mg (50 ml)

DAPI

Fluorescence dye for staining of chromosomes

236 276

10 mg

Anti-DigoxigeninFluorescein

Fab Fragments from sheep

1 207 741

200 µg

Anti-DigoxigeninRhodamine

Fab Fragments from sheep

1 207 750

200 µg

Anti-DigoxigeninAMCA

Fab Fragments from sheep

1 533 878

200 µg

These D N A probes are plasmids containing human ! satellite D N As. C lones were isolated from human-hamster hybrid cell lines containing the respective human chromosomes as the only human compo-

nent. The fragment length distribution of the labeled probes shows a maximum at 200–500 bases. 1 µg probe is sufficient for 20–50 hybridizations on 24 x 24 mm coverslips.

Reagent DNA probes, human chromosome " 1 specific, digoxigenin-labeled " 1 specific, fluorescein-labeled " 1 + 5 + 19 specific, digoxigenin-labeled " 1 + 5 + 19 specific, fluorescein-labeled " 2 specific, digoxigenin-labeled " 2 specific, fluorescein-labeled " 3 specific, digoxigenin-labeled " 4 specific, digoxigenin-labeled " 6 specific, digoxigenin-labeled " 7 specific, digoxigenin-labeled " 7 specific, fluorescein-labeled " 8 specific, digoxigenin-labeled " 8 specific, fluorescein-labeled " 9 specific, digoxigenin-labeled " 10 specific, digoxigenin-labeled " 10 specific, fluorescein-labeled " 11 specific, digoxigenin-labeled " 11 specific, fluorescein-labeled " 12 specific, digoxigenin-labeled " 12 specific, fluorescein-labeled " 13 + 21 specific, digoxigenin-labeled " 14 + 22 specific, digoxigenin-labeled " 14 + 22 specific, fluorescein-labeled " 15 specific, digoxigenin-labeled " 16 specific, digoxigenin-labeled " 17 specific, digoxigenin-labeled " 17 specific, fluorescein-labeled " 18 specific, digoxigenin-labeled " 18 specific, fluorescein-labeled " 20 specific, digoxigenin-labeled " 20 specific, fluorescein-labeled " 22 specific, digoxigenin-labeled " X specific, digoxigenin-labeled " X specific, fluorescein-labeled " Y specific, digoxigenin-labeled " Y specific, fluorescein-labeled " Specific for all human chromosomes, digoxigenin-labeled " Specific for all human chromosomes, fluorescein-labeled

CONTENTS

INDEX

Cat. No.

Pack size

1558 765 1558 684 1690 507 1690 647 1666 479 1666 380 1690 515 1690 523 1690 531 1690 639 1694 375 1666 487 1666 398 1690 540 1690 558 1690 655 1690 566 1690 663 1690 574 1690 671 1690 582 1666 495 1666 401 1690 604 1699 091 1666 452 1666 371 1666 509 1666 428 1666 517 1666 436 1690 612 1666 525 1666 444 1558 196 1558 692 1558 757 1558 676

1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl) 1 µg (50 µl)

5

93

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

Fluorescence in situ hybridization of a repetitive DNA probe to human chromosomes in suspension D . C eleda1, 2, U . Bettag1, and C . C rem er1 1 2

5

I nstitute for A pplied Physics, U niv ersity of H eidelberg. I nstitute for H um an G enetics and A nthropology, U niv ersity of H eidelberg, G erm any.

Fluorescence in situ hybridization (FISH ) has found widespread application in the analysis of chromosomes and interphase nuclei fixed on slides (Anastasi et al., 1990; C remer et al., 1988, 1990; D ekken et al., 1990; D evilee et al., 1988; Kolluri et al., 1990; Lichter et al., 1991; Pinkel et al., 1988; Schardin et al., 1985; Wienberg et al., 1990).

metaphase chromosomes with satisfactory morphology and clear hybridization signals (Figure 1).

H owever, hybridization of specific D N A probes to isolated metaphase chromosomes in suspension offers a new approach to chromosome analysis and chromosome separation. Initial investigations were done on chromosomes obtained from a (C hinese hamster X human) hybrid cell line with biotinylated human genomic D N A as the probe (D udin et al., 1987, 1988; H ausmann et al., 1991).

Procedure

So far the technique for FISH in suspension has been a modification of FISH techniques used for metaphase chromosomes and interphase nuclei fixed on slides. Formamide (and to some extent dextran sulfate) are obligatory components of this method.

! 1 Label the D N A probe pU C 1.77 with

Thus, the technique requires a certain number of washing steps after hybridization. The washing steps for FISH in suspension, however, are based on centrifugal steps. These steps are responsible for a considerable reduction in the final amount of chromosomal material. Additionally, the existing method for FISH in suspension appears to favor an aggregation of the chromosomal material in suspension. We report here a hybridization technique which does not need formamide and dextran sulfate. As a model system, we used the repetitive specific human D N A probe pU C 1.77 (C ooke and H indley, 1979; Emmerich et al., 1989), labeled it with digoxigenin-11-dU TP by nick-translation, and hybridized it to metaphase chromosomes in suspension. These chromosomes were isolated by standard techniques from human lymphocytes. In preliminary experiments, this technique produced a large number of isolated 94

Adapting the same method and probe to metaphase spreads (Figure 2) allowed us to determine hybridization efficiency.

N ote: T he D N A probe pU C 1.77 is a clone of the plasm id v ector pU C 9 that contains a 1.77 k b hum an Eco R I fragm ent. T he inserted D N A w as isolated from hum an satellite D N A fraction I I / I I I . T his insert contains m ainly a tandem ly organiz ed repetitiv e sequence in the region q12 of chrom osom e 1 (C ook e and H indley, 1979; G osden et al., 1981). D igoxigenin-11-dU TP according to standard nick translation procedures (as described in C hapter 4 of this manual). 2 C ultivate human lymphocytes from ! peripheral blood. ! 3 Prepare chromosomes from the lymphocytes by standard techniques. 4 Resuspend the chromosomes and inter! phase nuclei in methanol/ acetic acid (3 :1) and store at –20°C . 5 Perform fluorescence detection with ! Anti-D IG -Fluorescein, Fab fragments as described (Lichter et al., 1990), with the following modifications: " Incubate for approx. 1 h at 37°C . " After the blocking step, do not use bovine serum albumin in the rest of the procedure. " Modify as necessary to allow FISH in suspension. 6 After labeling and FISH procedures, ! pipette 10 µl of the suspension on a slide for microscopic analysis. C ounterstain with 15 µM propidium iodide (PI) and 5 µM D API. ! 7 Analyze with an epifluorescence microscope (O rthoplan equipped with a Planapo oil 63 x objective and a 1.40 aperture, Leitz, Wetzlar, G ermany).

CONTENTS

INDEX

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

8 Photograph with, for instance, Fuji!

chrome P1600 D film at a final image magnification of about 630 x, using Leitz filter system “I 2/ 3” for detection of FITC fluorescence.

Results

#

Figure 1: FISH of digoxigenin-labeled DNA probe pUC 1.77 to isolated human chromosomes in suspension. For details of the hybridization procedure, see the text. The sites of hybridization on the chromosomes appear as yellowish-green “ spots” in the centromeric regions. Counterstaining was with propidium iodide (PI) and DAPI.

5 Figure 2: FISH of a human metaphase spread " according to the same hybridization technique used in Figure 1. In this case the pUC 1.77 DNA probe binds to major hybridization sites (q12 region of human chromosome 1) that appear as two large yellowish-green “ spots” on two of the largest chromosomes (large arrowheads). Additionally, a minor binding site of the DNA probe (chromosome 16; Gosden et al., 1981) is indicated by two minor yellowish-green “ spots” on two of the smaller chromosomes (small arrowheads).

CONTENTS

INDEX

95

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

Reagents available from Boehringer Mannheim for this procedure

*This product or the use of this product may be covered by one or more patents of Boehringer Mannheim GmbH, including the following: EP patent 0 649 909 (application pending).

Reagent

Description

Cat. No.

Pack size

DIG-Nick Translation Mix* for in situ probes

5 x conc. stabilized reaction buffer in 50% glycerol (v/v) and DNAPolymerase I, DNase I, 0.25 mM dATP, 0.25 mM dCTP, 0.25 dGTP, 0.17 mM dTTP and 0.08 mM DIG-11-dUTP

1745 816

160 µl

Anti-DigoxigeninFluorescein

Fab Fragments from sheep

1 207 741

200 µg

DAPI

Fluorescence dye for staining of chromosomes

236 276

10 mg

Acknowledgments We thank Prof. D r. T. C remer, Intitute of H uman G enetics and Anthropology, U niversity of H eidelberg, for providing the plasmid pU C 9 and the human lymphocyte preparations. The work was supported by the D eutsche Forschungsgemeinschaft.

References Anastasi, J.; Le Beau M. M.; Vardiman, J. W.; Westbrook, C. A. (1990) Detection of numerical chromosomal abnormalities in neoplastic hematopoietic cells by in situ hybridization with a chromosome-specific probe. Am. J. Pathol. 136, 131–139. Cooke, H. J.; Hindley, J. (1979) Cloning of human satellite III DNA: different components are in different chromosomes. Nucleic Acids Res. 10, 3177–3197.

5

Cremer, T.; Lichter, P.; Borden, J.; Ward, D. C.; Manuelidis, L. (1988) Detection of chromosome aberrations in metaphase and interphase tumor cells by in situ hybridization using chromosomespecific library probes. Hum. Genet. 80, 235–246. Cremer, T.; Popp, S.; Emmerich, P.; Lichter, P.; Cremer, C. (1990) Rapid metaphase and interphase detection of radiation-induced chromosome aberrations in human lymphocytes by chromosomal suppression in situ hybridization. Cytometry 11, 110–118. Dekken van, H.; Pizzolo, J. G.; Reuter, V. E.; Melamed, M. R. (1990) Cytogenetic analysis of human solid tumors by in situ hybridization with a set of 12 chromosome specific DNA probes. Cytogenet. Cell Genet. 54, 103–107. Devilee, P.; Thierry, R. F.; Kievits, T.; Kolluri, R.; Hopman, A. H. N.; Willard, H. F.; Pearson, P. L.; Cornellisse, C. J. (1988) Detection of chromosome aneuploidy in interphase nuclei from human primary breast tumors using chromosome-specific repetitive DNA probes. Cancer Res. 48, 5825–5830. Dudin, G.; Cremer, T.; Schardin, M.; Hausmann, M.; Bier, F.; Cremer, C. (1987) A method for nucleic acid hybridization to isolated chromosomes in suspension. Hum. Genet. 76, 290–292.

96

Dudin, G.; Steegmayer, E. W.; Vogt, P.; Schnitzer, H.; Diaz, E.; Howell, K. E.; Cremer, T.; Cremer, C. (1988) Sorting of chromosomes by magnetic separation. Human. Genet. 80, 111–116. Emmerich, P.; Loos, P.; Jauch, A.; Hopman, A. H. N.; Wiegant, J.; Higgins, M. J.; White, B. N.; van der Ploeg, M.; Cremer, C.; Cremer, T. (1989) Double in situ hybridization in combination with digital image analysis: a new approach to study interphase chromosome topography. Exp. Cell Res. 181, 126–149. Gosden, J. R.; Lawrie, S. S.; Cooke, H.J. (1981) A cloned repeated DNA sequence in human chromosome heteromorphisms. Cytogenet. Cell Genet. 29, 32–39. Hausmann, M.; Dudin, G.; Aten, J. A.; Heilig, R.; Diaz, E.; Cremer, C. (1991) Slit scan flow cytometry of isolated chromosomes following fluorescence hybridization: an approach of online screening for specific chromosomes and chromosome translocations. Z. Naturforsch. 46c, 433–441. Kolluri, R. V.; Manuelidis, L.; Cremer, T.; Sait, S.; Gezer, S.; Raza, A. (1990) Detection of monosomy 7 in interphase cells for patients with myeloid disorders. Am. J. Hermatol. 33 (2), 117–122. Lichter, P.; Boyle, A. L.; Cremer, T.: Ward, D. C. (1991) Analysis of genes and chromosomes by non-isotopic in situ hybridization. Genet. Anal. Techn. Appl. 8, 24–35. Lichter, P.; Tang, C. C.; Call, K.; Hermanson, G.; Evans, H. J.; Housman, D.; Ward, D. C. (1990) High resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones. Science 247, 64–69. Pinkel, D.; Landegent, J.; Collins, C.; Fuscoe, J.; Segraves, R.; Lucas, J.; Gray, J. W. (1988) Fluorescence in situ hybridization with human chromosome-specific libraries: detection of trisomy 21 and translocations of chromosome 4. Proc. Natl. Acad. Sci. USA 85, 9138–9142. Schardin, M.; Cremer, T.; Hager, H. D.; Lang, M. (1985) Specific staining of human chromosomes in Chinese hamster X human cell lines demonstrates interphase territories. Hum. Genet. 71, 281–287. Wienberg, J.; Jauch, A.; Stanyon, R.; Cremer, T. (1990) Molecular cytotaxonomy of primates by chromosomal in situ suppression hybridization. Genomics 8, 347–350.

CONTENTS

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

III. Hybridization and detection ! 1 Add 5 µl of the hybridization mixture

containing the digoxigenin-labeled probe D N A (from Procedure I) to the chromosome preparation and cover with a coverslip (18 x 18 mm). 2 Seal coverslip with rubber cement. ! ! 3 Incubate slides at the appropriate hybridization temperature between 50°C and 65°C (depending on the probe, AT-content, sequence homology, etc.) for 4–6 h (for single copy sequences) or for 1 h (for repetitive sequences). N ote: L onger incubations do not m ark edly increase the hybridiz ation signal! 4 Remove rubber cement, then wash off !

the coverslip in 2 x SSC for 2–5 min at room temperature . 5 Wash the slide in PBS for 2 min. Remove ! excess buffer from the slide by wiping with soft paper around the chromosome preparation. N ote: D o not let the preparation dry com pletely; this produces back ground. 6 Apply 5 µl of a 1:10 diluted solution of !

fluorescein- or rhodamine-labeled antiD IG antibody. The antibodies should be diluted in PBS containing 1 mg/ ml bovine serum albumin.

5

! 7 Incubate 30 min with anti-D IG anti-

body at 37°C under a coverslip.

8 Wash 5 min in PBS. ! ! 9 Blot off excess buffer with paper. 10 Finally, mount the chromosomal prepa!

ration in “glycerol-para-phenylenediamine-mixture” [1 mg p-phenylenediamine dissolved in 1 ml phosphate buffered saline (1 mM sodium phosphate, pH 8.0; 15 mM N aC l) containing 50% glycerol]. 11 Inspect with a fluorescence microscope ! with the appropriate set of filters. C omments: I nstead of antibodies labeled w ith fluorescent dyes, any other antibody conjugates (enz ym e conjugated, goldlabeled) can be used. I n the case of biotin-labeled D N A , streptav idin can be used instead of antibodies to detect the hybridiz ed D N A . I hav e tested a num ber of these possibilities, and all w ork v ery w ell (including gold-labeled anti-digoxigenin antibody follow ed by silv er-enhancem ent). H ow ev er, in m y experience, the m ost precise localiz ation is obtained w ith im m unofluorescence. I t seem s to m e that the sensitiv ity is also higher (or at least the signal/ back ground ratio is im prov ed) w ith fluorescently labeled antibodies. A dm ittedly, this m ight be a m atter of personal preference.

Results Figure 1: Double in situ hybridization of a polytene chromosome of Chironomus with " twodifferent clones from a chromosomal walk within the sex-determining region of Chironomus piger. The two !-clones contain genomic DNA fragments which are 60 kbp apart. One clone was labeled with digoxigenin and detected with rhodamine-labeled anti-DIG antibody (red); the second clone was labeled with biotin and detected with anti-biotin antibody and fluorescein-labeled secondary antibody (green). The red and green signals can be seen simultaneously if the fluorescence microscope is equipped with a filter system for simultaneous blue and green excitation (filter no. 513803, Leica, Wetzlar). For the hybridization, the two probes are mixed 1:1, and the detection is performed with mixed antibody solutions. The method allows a very clear orientation for a chromosomal walk. We were able to discriminate the location of two clones which were only approximately 30 kb apart.

98

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

#

Figure 2: Double hybridization of a single copy clone from the sex-determining region together with a highly repetitive DNA element. The single copy DNA fragment was labeled with digoxigenin and the repetitive element with biotin. The single copy DNA(red) hybridizes to a single band, while the repetitive element (green) produces signals over numerous chromosomal sites throughout the entire chromosome. Hybridization and detection of the hybridized DNA was essentially the same as in Figure 1.

References

#

Figure 3: Double in situ hybridization of biotinand digoxigenin-labeled DNA probes from two different “DNA puffs” in the salivary gland polytene chromosome C of Trichosia pubescens (Sciaridae, Diptera). The probes were hybridized simultaneously as described in Figure 1. The two sites of hybridization were detected with rhodamine-labeled anti-DIG and anti-biotin antibodies plus fluorescein-labeled secondary antibody. The two differentially labeled sites are so-called “ DNA puffs” , which are chromosomal regions with a developmentally regulated DNA amplification. The double in situ hybridization method allows very rapid mapping of the chromosomes, even when the banding structure of the chromosome is not analyzed in detail.

Langer-Safer, P. R.; Levine, M.; Ward, D. C. (1982) Immunological method for mapping genes on Drosophila polytene chromosomes. Proc. Natl. Acad. Sci. USA 79, 4381–4385. Schmidt, E. R.; Keyl, H.-G.; Hankeln, T. (1988) In situ localization of two haemoglobin gene clusters in the chromosomes of 13 species of Chironomus. Chromosoma (Berl.) 96, 353–359.

5

Reagents available from Boehringer Mannheim for this procedure Reagent

Cat. No.

Pack size

DIG-High Prime* , **, *** Complete reaction mixture for random primed labeling of DNA with DIG-dUTP 5x solution with: 1 mM dATP, dCTP, dGTP (each), 0.65 mM dTTP, 0.35 mM DIG-11-dUTP, alkali-labile; random primer mixture; 1 unit/µl Klenow enzyme labeling grade, in reaction buffer, 50% glycerol (v/v)

1 585 606

160 µl (40 reactions)

Biotin-High Prime**

Complete reaction mixture for random primed labeling of DNA with Biotin-dUTP 5x solution with: 1 mM dATP, dCTP, dGTP (each), 0.65 mM dTTP, 0.35 mM biotin-16-dUTP, random primer mixture, 1 unit/µl Klenow enzyme labeling grade, in reaction buffer, 50% glycerol (v/v)

1 585 649

100 µl (25 reactions)

RNase, DNase-free

RNase, DNase-free, can be used directly without prior heat treatment in any DNA isolation technique

1119 915

500 µg (1 ml)

Anti-DigoxigeninRhodamine

Fab Fragments from sheep

1 207 750

200 µg

CONTENTS

Description

***EP Patent 0 324 474 granted to and EP Patent application 0 371 262 pending for Boehringer Mannheim GmbH. ***This product or the use of this product may be covered by one or more patents of Boehringer Mannheim GmbH, including the following: EP patent 0 649 909 (application pending). ***Licensed by Institute Pasteur.

INDEX

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

A simplified and efficient protocol for nonradioactive in situ hybridization to polytene chromosomes with a DIG-labeled DNA probe Prof. D r. E. R . Schm idt, I nstitute for G enetics, Johannes G utenberg-U niv ersity of M ainz , G erm any.

The protocol given here is a derivative of several published methods (Langer-Safer et al., 1982; Schmidt et al., 1988) with some minor modifications that make the method of in situ hybridization easier, faster, more reliable, and available to anyone who can operate a microscope.

I. Labeling the hybridization probe For best labeling results, follow the random primed D N A labeling procedure (Procedure IA) in C hapter 4 of this manual. We suggest a modification of this procedure as described below: ! 1 U se 0.5–1 µg linearized D N A in 16 µl

redistilled H 2O .

2 D enature in a boiling water bath for !

10 min and chill on ice.

! 3 Add 4 µl D IG -H igh Prime reaction

mix.

4 Mix, centrifuge and incubate either 2 h at !

37° C or overnight at room temperature. 5 Stop the labeling procedure by heating ! in boiling water for 10 min. 6 Add water and 20 x SSC to give a final ! volume of 100 µl with a final concentration of 5 x SSC . N ote: I f a larger num ber of preparations is to be hybridiz ed, increase the v olum e to 200 µl. ! 7 Add 1 volume of 10% SDS to 100 volumes

of the mixture in Step 6 (i.e., 1 µl SDS to 100 µl of mixture) to give a final concentration of 0.1% SDS. 8 The mixture is ready for hybridization ! and can be stored at –20°C for at least several years without any significant decrease in hybridization efficiency. C omment: I n m y experience, it is absolutely unnecessary to rem ov e unincorporated dN T Ps from the m ixture before using the labeled probe. O n the contrary, purification steps lead to the loss of hybridiz able probe D N A and thus to a decrease in hybridiz ation efficiency.

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II. Preparation and denaturation of polytene chromosomes from Drosophila, Chironomus, or other species Squash larval salivary glands in 40% acetic acid according to standard procedure. For long term storage, place slides with squashed chromosomes in 100% 2-propanol at –20°C. Prior to the in situ hybridization, denature the chromosomal D N A according to this procedure: ! 1 Rehydrate the slides by incubating them,

for 2 min each, in solutions containing decreasing amounts of ethanol: 70% ethanol, 50% ethanol, 30% ethanol, 0.1 x SSC –2 x SSC . 2 D igest with RN ase if necessary. ! N ote: T his is usually not necessary. ! 3 For better preservation of the chromo-

somes, include a “heat stabilization” step by incubating slides in 2 x SSC for at least 30 min at 80°C . 4 Incubate the slides for 90 s in 0.1 N ! N aO H , at room temperature. 5 Wash slides for 30 s in 2 x SSC . ! 6 D ehydrate slides by incubating them, ! for 2 min each, in solutions containing increasing amounts of ethanol: 30% ethanol, 50% ethanol, 70% ethanol, 95% ethanol. ! 7 Air dry the slides for 5 min. The preparations are then ready for hybridization. C omment: I f “denatured” chrom osom e preparations are to be stored for a long tim e, store them in 95% ethanol or 2-propanol at –20° C .

5

97

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

Multiple-target DNA in situ hybridization with enzyme-based cytochemical detection systems E. J. M . Speel, F. C . S. R am aek ers, and A . H . N . H opm an, D epartm ent of M olecular C ell Biology & G enetics, U niv ersity of L im burg, M aastricht, T he N etherlands.

Fluorescence in situ hybridization (ISH ) is widely utilized because of its high sensitivity, resolution, and ability to detect multiple cellular nucleic acid sequences in different colors. Fluorescence ISH , however, has disadvantages, such as: ! Fluorescence signals fade when they are exposed to light. ! Autofluorescence in, e.g., tissue sections can interfere with target analysis. H ere we outline a multicolor enzyme-based cytochemical detection protocol for nucleic acids in situ. This protocol produces permanent cell preparations with non-diffusible, nonfading reaction products. The reaction products can be analyzed with brightfield (Speel et al., 1994a), reflection-contrast (Speel et al., 1993), or, in one case (alkaline phosphatase-Fast Red reaction), fluorescence microscopy (Speel et al., 1992). We show examples of single- and multipletarget ISH experiments on standard human lymphocyte metaphase spreads, as well as on interphase cell preparations. These results demonstrate the potential of this detection methodology for metaphase and interphase cytogenetics, e.g., for studying chromosome aberrations in different cell types (Martini et al., 1995). In addition, this methodology can be applied in the area of pathology, since the universal detection protocol described here can be combined with other sample preparation procedures, e.g., for tissue sections (H opman et al., 1991, 1992).

5

The procedures given below are modifications of previously published procedures (Speel et al., 1992, 1993, 1994a, 1994b).

I. Cell preparations Lym phocytes: Prepare chromosomes from peripheral blood lymphocytes by standard methods. Fix in methanol:acetic acid (3:1, v/ v), and drop chromosomes onto glass slides that have been cleaned with a 1:1 mix of ethanol and ether. C ultured cells: Make preparations from cultured normal diploid cells or tumor cell lines by one of the following methods:

100

! Ethanol suspension: Trypsinize cells (if

necessary), harvest, wash in PBS, fix in cold 70% ethanol (–20°C ), and drop onto glass slides that have been coated with poly-L-lysine. ! Slide and coverslip preparations: G row cells on glass slides or coverslips. Fix in cold methanol (–20° C ) for 5 s, then in cold acetone (4° C ) for 3 x 5 s. Air dry samples and store at –20°C . N ote: A lternativ ely, use other fixativ es for the slide and cov erslip preparations. ! C ytospins: C ytospin floating cells onto glass slides at 1000 rpm for 5 min. Air dry samples for 1 h at room temperature. Fix and store as with slide and coverslip preparations above.

II. Cell processing " 1 D ecide whether samples need to be

treated with RN ase. Then do one of the following: ! I f the cells need RN ase, go to Step 2. ! I f the cells do not need RN ase, go to Step 3. 2 Treat slides with RN ase as follows: " ! O verlay each sample with 100 µl RN ase solution (100 µg/ ml RN ase A in 2 x SSC ) and a coverslip. ! Incubate cell samples for 1 h at 37° C . ! Remove coverslip and wash samples 3 x 5 min with 2 x SSC . ! G o to Step 3. " 3 Treat slides with pepsin as follows: ! O verlay each sample with 100 µl pepsin solution (50–100 µg/ ml pepsin in 10 mM H C l) and a coverslip. ! Incubate cell samples for 10–20 min at 37°C . ! Wash samples as follows: – 2 min with 10 mM H C l – 2 x 5 min with PBS 4 Post-fix samples as follows: " ! Incubate samples with PBS containing 1% (para)formaldehyde for either 20 min at 4°C or 10 min at room temperature. ! Wash slides 2 x 5 min with PBS. ! D ehydrate samples by passing slides through a series of ethanol solutions (70% , then 96% , then 100% ethanol), incubating 10–60 s in each solution.

CONTENTS

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

III. Probe preparation " 1 Label the D N A probes (containing either

repetitive or unique sequences) with Biotin-, D igoxigenin-, or FluoresceindU TP according to the nick translation procedure in C hapter 4 of this manual. 2 Just before use, prepare hybridization " buffer containing: ! 50% or 60% formamide. ! 10% dextran sulfate. ! 2 x SSC . ! 0.2 µg/ µl sonicated herring sperm D N A. ! 0.2 µg/ µl yeast tRN A. ! 1–2 ng labeled probe D N A/ µl hybridization buffer [if the probe contains unique or highly repetitive (e.g. centromere probe) sequences] or 2–4 ng labeled probe D N A/ µl hybridization buffer, together with an excess (100–1000 fold) of total human D N A or C o t-1 D N A [if the probe contains repetitive (e.g. A lu) elements]. " 3 Perform ISH by one of the following methods: ! I f the probe contains unique or highly repetitive (e.g. centromere probe) sequences, follow procedure IVA. ! I f the probe contains repetitive (e.g. A lu) elements, follow procedure IVB. ! For multiple-target in situ hybridization, prepare D N A probes labeled with different haptens (biotin, digoxigenin, or fluorescein), mix them together, and follow either Procedure IVA or Procedure IVB (depending on the nature of the probes).

CONTENTS

INDEX

IV. Multiple-target in situ hybridization (ISH) IVA. ISHwith simultaneous probe and target denaturation [for probes with unique or highly repetitive (e.g., centromere probe) sequences] " 1 O n each sample, place 10 µl of hybridi-

zation buffer containing labeled probe D N A (prepared as in Procedure III, Step 2). 2 C over sample with a 20 x 20 mm cover" slip and (if you wish) seal the coverslip to the slide with rubber cement. " 3 D enature probe and cellular D N A simultaneously by placing slides at 70°– 75°C for 3–5 min on the bottom of a metal box. 4 Incubate hybridization samples over" night at 37°C . 5 G o to Procedure V. " IVB. ISH with separate probe and target denaturation [for probes with repetitive (e.g. Alu) elements] " 1 Incubate the probe mixture (labeled

probe D N A, human or C o t-1 D N A, hybridization buffer; prepared as in Procedure III, Step 2) for 5 min at 75°C . 2 C hill the denatured probe mixture on " ice. " 3 Incubate the denatured probe mixture for 1–4 h at 37°C to pre-anneal the repetitive sequences in the mixture. 4 D enature the cell samples as follows: " ! O verlay the cell samples with 70% formamide in 2 x SSC . ! Incubate the slides for 2 min at 70°C to denature the cells. ! D ehydrate the cell samples in a series of chilled (–20° C ) ethanol solutions (70% , then 96% , then 100% ethanol), incubating them for 5 min in each solution. ! Air dry the samples. 5 O n each denatured cell sample (from " Step 4), place 10 µl denatured, preannealed probe mixture (from Step 3). 6 Incubate hybridization samples over" night at 37°C . " 7 G o to Procedure V.

5

101

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

6 Repeat Step 5 with the next incubation "

V. Post-hybridization washes " 1 Perform the following stringent washes

of the samples (from either Procedure IVA or Procedure IVB) at 42°C : ! 2 x 5 min with 2 x SSC containing 50% (or 60% ) formamide and 0.05% Tween ® 20. ! 2 x 5 min with 2 x SSC . 2 D epending upon the nature of the " probe, do one of the following: ! I f the probe contains repetitive (e.g. A lu) elements, wash the samples 2 x 5 min at 60°C with 0.01 x SSC . ! I f the probe does not contain repetitive elements, skip this step.

VI. Enzyme-based cytochemical detection

in the detection system (Table 1) until all incubations are complete. " 7 After all incubations in the detection system are complete, wash samples 5 min with PBS. 8 Visualize according to one of the proce" dures in Section VII. " 9 If the detection procedure uses the same enzyme (peroxidase or alkaline phosphatase) to detect two different probes, do the following: ! Inactivate the enzyme on the first detecting molecule by incubating the sample for 10 min at room temperature with 10 mM H C l. ! Repeat Steps 5–8 with a second detection system that recognizes the sec-ond probe (Speel et al., 1994b).

VIA. Single color detection Table 1: Frequently used detection ! Probe systems for enzyme-based in situ label hybridization.

5

1 Abbreviations used: Ab, antibody; ABC, avidin-biotinylated enzyme (horseradish peroxidase or alkaline phosphatase) complex; E, enzyme (horseradish peroxidase or alkaline phosphatase). 2 Hapten = biotin, digoxigenin, FITC, or DNP. 3 Anti-hapten Ab raised in another species (e.g., rabbit, goat, swine) can also be used as primary Ab in probe detection schemes.

102

Biotin Biotin H apten 2 H apten H apten H apten H apten

Incubation 2

1 Avidin-E 1 Avidin-E Anti-hapten Ab-E Mouse3 anti-hapten Ab Mouse anti-hapten Ab Mouse anti-hapten Ab Mouse anti-hapten Ab

3

Biotinylated anti-avidin Ab

Avidin-E

Anti-mouse Ab-E Rabbit anti-mouse Ab-E Biotinylated anti-mouse Ab D IG -labeled anti-mouse Ab

Anti-rabbit Ab-E ABC Anti-D IG Ab-E

" 1 Wash samples briefly with 4 x SSC

containing 0.05% Tween ® 20. 2 Incubate samples for 10 min at 37° C " with 4 x SSC containing 5% nonfat dry milk. " 3 C hoose an appropriate enzyme-based detection system (Table 1). 4 D ilute detecting molecules as follows: " ! D ilute avidin conjugates in 4 x SSC containing 5% nonfat dry milk. ! D ilute antibody conjugates in PBS containing 2–5% normal serum and 0.05% Tween ® 20. 5 For the first incubation in the detection " system (Table 1), do the following: ! Incubate samples with diluted detecting molecule for 30 min at 37°C . ! Wash samples 2 x 5 min in the appropriate wash buffer (4 x SSC for avidin; PBS for antibodies) containing 0.05% Tween ® 20.

VIB. Multiple target, multicolor detection

To detect multiple probes labeled with different haptens, use a combination of detection systems. For example, Table 2 outlines a protocol for triple-target in situ hybridization. For protocols with doubletarget in situ hybridizations, see H opman et al. (1986), Emmerich et al. (1989), Mullink et al. (1989), H errington et al. (1989), Kerstens et al. (1994), and Speel et al. (1995). N ote: A lternativ ely, if tw o peroxidase or phosphatase reactions are used in the detection protocol (Speel et al., 1994b), inactiv ate the enz ym e after the first detection reaction in 10 m M H C l for 10 m in at room tem perature, then perform the second detection reaction (as in Procedure V I A abov e).

CONTENTS

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

D etection step

Incubation time2

Incubation temperature

20 min

37°C

2. Visualize PO by Procedure VIIA (PO -D AB, brown signal)

5 min

37° C

3. Inactivate residual AvPO with 10 mM H C l.

10 min

RT

4. D etect digoxigenin and FITC with MAD IG and RAFITC (each diluted 1:2000)

30 min

37°C

5. D etect anti-digoxigenin and anti-FITC with G AMAPase (diluted 1:25) and SWARPO (diluted 1:100)

30 min

37°C

6. Visualize APase activity by Procedure VIIC (APase-Fast Red, red signal)

5–10 min

37°C

7. Visualize PO activity by Procedure VIIB (PO -TMB, green signal)

1–2 min

37° C

8. C ounterstain with hematoxylin

1 sec

RT

9. Air dry

10 min

RT

10 min

37°C

1. D etect biotin with

AvPO 1

10. Embed in a protein

(diluted 1:50)

matrix3

# Table 2: Detection protocol for triple-target in situ hybridization with a biotin-, digoxigenin-, and a FITC-labeled probe. 1 Abbreviations used: Ab, antibody; APase, alkaline phosphatase; AvPO, PO-conjugated avidin (Vector); DAB, diaminobenzidine; GAMAPase, APase-conjugated goat anti-mouse Ab (DAKO); MADIG, mouse anti-digoxigenin Ab; PO, horseradish peroxidase; RAFITC, rabbit anti-FITC Ab (DAKO); RT, room temperature; SWARPO, PO-conjugated swine anti-rabbit Ab (DAKO). 2 For details of detection reactions, see Procedure VIA. For details of visualization reactions, see Procedures VIIA–VIID. 3 For details of protein matrix, see Procedure VIII.

VII. Visualization Enzyme label

Substrate

Precipitate colors in microscopy Brightfield Reflection-contrast Fluorescence

PO 2

H 2O 2/ D AB H 2O 2/ TMB

Brown G reen

White Pink/ Red

– –

APase

N -ASMX-P/ FR BC IP/ N BT

Red Purple

Yellow Yellow/ O range

Red –

D epending upon the type of microscopy to be used for analysis (Table 3) and the detecting molecule, choose one of the following procedures to visualize the hybrids. In our hands, each of these procedures is optimal for in situ hybridization. VIIA. Horseradish peroxidasediaminobenzidine (PO-DAB) " 1 Mix color reagent just before use: ! 1 ml 3,3-diaminobenzidine tetra-

chloride (D AB; Sigma) stock (5 mg D AB/ ml PBS). ! 9 ml PBS containing 0.1 M imidazole, pH 7.6. ! 10 µl 30% H 2O 2. 2 O verlay each sample with 100 µl color " reagent and a coverslip. " 3 Incubate samples for 5–15 min at 37°C . 4 Wash samples 3 x 5 min with PBS and (if " you wish) dehydrate them. 5 C overslip with an aqueous or organic " mounting medium.

CONTENTS

INDEX

VIIB. Horseradish peroxidasetetramethylbenzidine (PO-TMB) " 1 D issolve 100 mg sodium tungstate

(Sigma) in 7.5 ml 100 mM citratephosphate buffer (pH 5.1). Adjust the pH of the tungstate solution to pH 5.0–5.5 with 37% H C l. 2 Just before use, dissolve 20 mg dioctyl " sodium sulfosuccinate (Sigma) and 6 mg 3,3',5,5'-tetramethylbenzidine (TMB, Sigma) in 2.5 ml 100% ethanol at 80°C . " 3 Prepare 10 ml color reagent by combining the tungstate solution (Step 1), the TMB solution (Step 2), and 10 µl 30% H 2O 2. 4 O verlay each sample with 100 µl color " reagent and a coverslip. 5 Incubate samples for 1–2 min at 37°C . " 6 Wash samples 3 x 1 min with ice-cold " 100 mM phosphate buffer (pH 6.0) and (if you wish) dehydrate them. " 7 C overslip with an organic mounting medium or immersion oil.

# Table 3: Enzyme reaction

protocols and colors in different types of light microscopy1.

1 Other enzyme reactions that have been used for ISH are described in Speel et al. (1993, 1995). 2 Abbreviations used: APase, alkaline phosphatase; BCIP, bromochloro-indolyl phosphate; DAB, diaminobenzidine; FR, Fast Red TR; N-ASMX-P, naphthol-ASMXphosphate; NBT, nitroblue tetrazolium; PO, horseradish peroxidase; TMB, tetramethylbenzidine.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

VIIC. Alkaline phosphatase-Fast Red (APase-Fast Red)

Results

" 1 Mix color reagent just before use: ! 4 ml TM buffer [200 mM Tris-H C l

(pH 8.5), 10 mM MgC l2] containing 5% polyvinyl alcohol (PVA, MW 40,000; Sigma). ! 250 µl TM buffer containing 1 mg naphthol-ASMX-phosphate (Sigma). ! 750 µl TM buffer containing 5 mg Fast Red TR salt (Sigma). 2 O verlay each sample with 100 µl color " reagent and a coverslip. " 3 Incubate samples for 5–15 min at 37°C . 4 Wash samples 3 x 5 min with PBS. " 5 C overslip with an aqueous mounting " medium. VIID. Alkaline phosphatase-bromochloroindolyl phosphate (APase-BCIP/NBT)

Follow the standard procedure given in C hapter 2 of this manual.

VIII. Embedding and light microscopy " 1 Prepare samples for microscopy by

doing either of the following: ! I f samples require a single mounting medium, embed stained samples as described in Procedure VII. ! I f multiple precipitation reactions would require different (aqueous or organic) embedding mediums, apply instead a protein embedding layer by smearing 50 µl of a 1:1 mixture of BSA solution (40 mg/ ml in deionized H 2O ) and 4% formaldehyde onto the slides. Air dry for 10 min at 37°C . N ote: T his protein em bedding layer can be used to prev ent solubiliz ation of enz ym e precipitates during all types of light m icroscopy (Speel et al., 1993, 1994a). 2 Perform brightfield, fluorescence and " reflection contrast microscopy as described in the literature (Speel et al., 1992, 1993, 1994b; Cornelese-Ten Velde et al., 1989).

5

104

$

Figure 1: Single-target ISH on a normal human lymphocyte metaphase spread with a peroxidase detection system. The probe was a biotinylated cosmid that recognizes 40 kb of chromosome 11q23. The detection system included monoclonal anti-biotin Ab (DAKO), rabbit anti-mouse Ab-PO, and the PO-DABreaction. The sample was counterstained with hematoxylin and viewed by brightfield microscopy.

$

Figure 2: Double-target ISH on normal human umbilical vein endothelial cells with brightfield viewing. The centromere of chromosome 1 (brown) was detected with a biotinylated probe; that of chromosome 7 (red), with a digoxigenin-labeled probe. The detection steps included (1) avidin-PO, (2) monoclonal anti-digoxigenin Ab and rabbit antimouse Ab-APase, (3) the APase-Fast Red reaction, and (4) the PO-DAB reaction. The sample was counterstained with hematoxylin and viewed by brightfield microscopy.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

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Figure 3: Same ISH experiment as in Figure 1, but with a fluorescent alkaline phosphatase detection system. The detection system for the biotinylated cosmid probe included monoclonal anti-biotin, horse anti-mouse Ab-biotin, avidinbiotinylated APase complex, and the APase-Fast Red reaction (Speel et al., 1994a). The sample was counterstained with Thiazole Orange (Molecular Probes) and viewed by fluorescence microscopy.

$

Figure 4: Triple-target ISHon a normal human lymphocyte metaphase spread. Probes specific for the centromeres of chromosomes 1 (brown), 7 (red), and 17 (green) were labeled with biotin, digoxigenin, and FITC, respectively. Detection, counterstaining, and embedding in a BSA protein layer was as outlined in the text (Table 2). The sample was viewed by brightfield microscopy.

5

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Figure 5: Triple-target ISH on human bladder tumor cell line T24. The probes and experimental procedures were the same as in Figure 4.

Reprinted from Speel, E. J. M.; Kamps, M.; Bonnet, J.; Ramaekers, F. C. S.; Hopman, A. H. N.; (1993) Multicolor preparations for in situ hybridization using precipitating enzyme cytochemistry in combination with reflection contrast microscopy. Histochemistry 100, 357–366 with permission of Springer-Verlag GmbH & Co. KG.

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Figure 6: Double-target ISH on a normal human lymphocyte metaphase spread with reflection contrast viewing. The centromere of chromosome1 (yellow) was detected with a biotinylated probe; that of chromosome 17 (white), with a digoxigeninlabeled probe. The detection steps included (1) monoclonal anti-biotin Ab and rabbit anti-digoxigenin Ab, (2) horse anti-mouse Ab-biotin and swine anti-rabbit Ab-PO, (3) streptavidin-biotinylated APase complex, (4) the APase-Fast Red reaction, and (5) the PO-DAB reaction (Speel et al., 1993). The sample was not counterstained, but was embedded in a BSA protein layer and viewed by reflection contrast microscopy.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

References Cornelese-Ten Velde, I.; Wiegant, J.; Tanke, H. J.; Ploem, J. S. (1989) Improved detection and quantification of the (immuno)peroxidase product using reflection contrast microscopy. Histochemistry 92, 153–160.

Martini. E.; Speel, E. J. M.; Geraedts, J. P. M.; Ramaekers, F. C. S.; Hopman, A. H. N. (1995) Application of different in situ hybridization detection methods for human sperm analysis. Hum. Reprod. 10, 855–861.

Emmerich, P.; Loos, P.; Jauch, A.; Hopman, A. H. N.; Wiegant, J.; Higgins, M. J.; White, B. N.; Van der Ploeg, M.; Cremer, C.; Cremer, T. (1989) Double in situ hybridization in combination with digital image analysis: a new approach to study interphase chromosome topography. Exp. Cell Res. 181, 126–140.

Mullink, H.; Walboomers, J. M. M.; Raap, A. K.; Meyer, C. J. L. M. (1989) Two colour DNA in situ hybridization for the detection of two viral genomes using non-radioactive probes. Histochemistry 91, 195–198.

Herrington, C. S.; Burns, J.; Graham, A. K.; Bhatt, B.; McGee, J. O’D. (1989) Interphase cytogenetics using biotin and digoxygenin labeled probes II: simultaneous differential detection of human and papilloma virus nucleic acids in individual nuclei. J. Clin. Pathol. 42, 601–606. Hopman, A. H. N.; Wiegant, J.; Raap, A. K.; Landegent, J. E.; Van der Ploeg, M.; Van Duijn, P. (1986) Bi-color detection of two target DNAs by non-radioactive in situ hybridization. Histochemistry 85, 1–4. Hopman, A. H. N.; Van Hooren, E.; Van de Kaa, C. A.; Vooijs, G. P.; Ramaekers, F. C. S. (1991) Detection of numerical chromosome aberrations using in situ hybridization in paraffin sections of routinely processed bladder cancers. Modern Pathol. 4, 503–513. Hopman, A. H. N.; Poddighe, P. J.; Moesker, O.; Ramaekers, F. C. S. (1992) Interphase cytogenetics: an approach to the detection of genetic aberrations in tumours. In: McGee, J. O’D.; Herrington, C. S. (Eds.) Diagnostic Molecular Pathology, A Practical Approach, 1st ed. New York: IRL Press, 141–167.

5

Kerstens, H. M. J.; Poddighe, P. J.; Hanselaar, A. G. J. M. (1994) Double-target in situ hybridization in brightfield microscopy. J. Histochem. Cytochem. 42, 1071–1077.

106

Speel, E. J. M.; Schutte, B.; Wiegant, J.; Ramaekers, F. C. S.; Hopman, A. H. N. (1992) A novel fluorescence detection method for in situ hybridization, based on the alkaline phosphatase-Fast Red reaction. J. Histochem. Cytochem. 40, 1299–1308. Speel, E. J. M.; Kamps, M.; Bonnet, J.; Ramaekers, F. C. S.; Hopman, A. H. N. (1993) Multicolour preparations for in situ hybridization using precipitating enzyme cytochemistry in combination with reflection contrast microscopy. Histochemistry 100, 357–366. Speel, E. J. M.; Herbergs, J.; Ramaekers, F. C. S.; Hopman, A. H. N. (1994a) Combined immunocytochemistry and fluorescence in situ hybridization for simultaneous tricolor detection of cell cycle, genomic, and phenotypic parameters of tumor cells. J. Histochem. Cytochem. 42, 961–966. Speel, E. J. M.; Jansen, M. P. H. M.; Ramaekers, F. C. S.; Hopman, A. H. N. (1994b) A novel triplecolor detection procedure for brightfield microscopy, combining in situ hybridization with immuocytochemistry. J. Histochem. Cytochem. 42, 1299–1307. Speel, E. J. M.; Ramaekers, F. C. S.; Hopman, A. H. N. (1995) Detection systems for in situ hybridization, and the combination with immunocytochemistry. Histochem. J., in press.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

Reagents available from Boehringer Mannheim for this procedure Reagent

Description

Cat. No.

Pack size

RNase A

Dry powder

109 142 109 169

25 mg 100 mg

Pepsin

Aspartic endopeptidase with broad specificity

108 057 1 693 387

1g 5g

Digoxigenin-11-dUTP, alkali-stable

Tetralithium salt, 1 mM solution

1 093 088

25 nmol (25 µl) 125 nmol (125 µl) 5x125 nmol (5x125 µl)

1 558 706 1 570 013 Fluorescein-12-dUTP

Tetralithium salt, 1 mM solution

1 373 242

25 nmol (25 µl)

Tetramethylrhodamine-6-dUTP

Tetralithium salt, 1 mM solution

1 534 378

25 nmol (25 µl)

AMCA-6-dUTP

Tetralithium salt, 1 mM solution

1 534 386

25 nmol (25 µl)

Biotin-16-dUTP

Tetralithium salt, 1 mM solution

1 093 070

50 nmol (50 µl)

DNase I

Lyophilizate

104 159

100 mg

DNA Polymerase I

Nick Translation Grade

104 485 104 493

500 units 1000 units

tRNA

From baker’s yeast

109 495 109 509

100 mg 500 mg

DNA, Cot-1, human

Solution in 10 mM Tris-HCl, 1 mM EDTA, pH 7.4, Cot-1 DNA is used in chromosomal in situ suppression (CISS).

1 581 074

500 µg (500 µl)

1 332 465

5x10 ml

Tween® 20 Anti-Digoxigenin

Clone 1.71.256, mouse IgG 1, !

1 333 062

100 µg

Anti-Mouse IgDigoxigenin

F(ab)2 fragment

1 214 624

200 µg

Anti-Digoxigenin-POD

Fab fragments from sheep

1 207 733

150 U

Anti-Digoxigenin-AP

Fab fragments from sheep

1 093 274

150 U

NBT/BCIP, (stock solution)

Solution of 18.75 mg/ml nitro-blue tetrazolium chloride and 9.4 mg/ml 5-bromo-4-chloro-3-indolyl phosphate, toluidine-salt in 67% DMSO (v/v)

1 681 451

8 ml

Tris

Powder

127 434 708 968 708 976

100 g 500 g 1 kg

BSA

Highest quality, lyophilizate

238 031 238 040

1g 10 g

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107

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

DNA in situ hybridization with an alkaline phosphatase-based fluorescent detection system D r. G . Sagner, R esearch L aboratories, Boehringer M annheim G m bH , Penz berg, G erm any.

We describe here a high sensitivity indirect detection procedure for D IG -labeled hybridization probes. The procedure uses the components of the H N PP Fluorescent Detection Set to form a fluorescent precipitate of H N PP (2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate) and Fast Red TR at the site of hybridization. This procedure can be used to detect single copy sequences as small as 1 kb on human metaphase chromosomes.

I. In situ hybridization with DIG-labeled probes Prepare chromosomal spreads, label hybridization probes with digoxigenin (D IG ), hybridize probes to chromosomes, and perform posthybridization washes of the sample according to standard procedures, e.g. those listed in C hapters 4 and 5 of this manual.

5

II. Detection of DIG-labeled probes " 1 Prepare a fresh 1:500 dilution of alkaline

phosphatase-conjugated sheep anti-D IG antibody in blocking buffer [100 mM Tris-H C l, 150 mM N aC l, 0.5% blocking reagent; pH 7.5 (20°C )]. N ote: D o not store the diluted antibody conjugate m ore than 12 h at 4° C .

108

! Prepare Fast Red TR stock solution

by dissolving 5 mg Fast Red TR (vial 2) in 200 µl distilled H 2O . ! Mix 10 µl H N PP stock solution (premixed, vial 1), 10 ml Fast Red TR stock solution, and 1 ml detection buffer [100 mM Tris-H C l, 100 mM N aC l, 10 mM MgC l2; pH 8.0 (20° C )]. ! Filter the H N PP/ Fast Red TR mix through a 0.2 µm nylon filter. C aution: T he Fast R ed T R stock solution should be no m ore than 4 w eek s old. T he H N PP/ Fast R ed T R m ix should be no m ore than a few days old. A lw ays filter the m ix just before use. 6 C over each sample with 100 µl filtered "

H N PP/ Fast Red TR mix and a coverslip, then incubate slides for 30 min at room temperature. " 7 Wash the slides 1x at room temperature with washing buffer [100 mM Tris-H C l, 150 mM N aC l, 0.05% Tween ® 20; pH 7.5 (20° C )]. 8 Repeat Steps 6 and 7 three times. " N ote: I f you are detecting abundant or m ultiple copy sequences, sk ip Step 8. O ne incubation is enough. " 9 Wash the slides for 10 min with distilled

H 2O .

III. Fluorescence microscopy

2 Pipette 100 µl of diluted anti-D IG "

" 1 In a C oplin jar, counterstain slides with

antibody conjugate onto each slide and add a coverslip. " 3 Incubate slides for 1 h at 37°C in a moist chamber. 4 Wash the slides at room temperature as " follows: ! 3 x 10 min with washing buffer [100 mM Tris-H C l, 150 mM N aC l, 0.05% Tween ® 20; pH 7.5 (20° C )]. ! 2 x 10 min with detection buffer [100 mM Tris-H C l,100 mM N aC l, 10 mM MgC l2; pH 8.0 (20° C )]. 5 Prepare fresh H N PP/ Fast Red TR mix " as follows: N ote: N um bered v ials are com ponents of the H N PP Fluorescent D etection Set.

50 ml D API solution (100 ng D API/ ml PBS) for 5 min in the dark at room temperature. 2 Wash slides under running water for " 2–3 min. " 3 Air dry slides in the dark. 4 Add 20 µl D ABC O antifading solution " (PBS containing 50% glycerol and 2% D ABC O ) to each slide. 5 C over each slide with a 24 x 24 mm " coverslip. N ote: For long term storage of the slides, seal the edges of the cov erslip w ith nail polish and store in the dark at –20° C . 6 View the slides by fluorescence micros"

copy, using appropriate filters.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es

N ote: T he m axim um em ission w av elength of dephosphorylated H N PP/ Fast R ed T R is 562 nm . H ow ev er, since the em ission peak is broad (540–590 nm ), you m ay use the filter sets for either fluorescein or rhodam ine to analyz e the H N PP/ Fast R ed T R product.

Results The fluorescent signal obtained with the H N PP/ Fast Red procedure described above is more intense than a direct signal from fluorescein. In a comparison experiment (Figure 1), the H N PP/ Fast Red reaction product was visible after a 70-fold shorter exposure (0.2 sec exposure vs. 15 s exposure) than the fluorescein signal. Even when the H N PP/ Fast Red signal is viewed under a less than optimal filter (Figure 2), the fluorescence is clearly visible.

# Figure 1: Comparison of fluorescein and HNPP signal intensity. A DIG-labeled painting

probe specific for human chromosome 1 was hybridized to metaphase chromosomal spreads. The DIG-labeled probe was detected with (Panel a) anti-DIG-fluorescein conjugate or (Panel b) anti-DIG-alkaline phosphate conjugate and the HNPP/Fast Red detection reaction described in the text. The fluorescein signal (Panel a) required a 15 s exposure while the HNPP/Fast Red signal (Panel b) required a 0.2 s exposure. Fluorescent images were pseudocolored with a CCD camera and a digital imaging system. Chromosomes were counterstained with DAPI.

a

# Figure 2: HNPP/Fast Red

b

Reagents available from Boehringer Mannheim for this procedure Reagent

Description

Cat. No.

Pack size

Tris

Powder

127 434 708 968 708 976

100 g 500 g 1 kg

Blocking Reagent

Powder

1 096 176

50 g

Anti-Digoxigenin-AP

Fab Fragments from sheep

1 093 274

150 U

1 332 465

5x10 ml

1758 888

Set for 500 FISH reactions

236 276

10 mg

Tween® 20 HNPP Fluorescent Detection Set

For sensitive fluorescent detection of nonradioactively labeled nucleic acids in fluorescence in situ hybridization (FISH) and membrane hybridization

DAPI

Fluorescence dye for staining of chromosomes

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fluorescence viewed with a filter for DAPI. A DIG-labeled painting probe specific for human chromosome 1 was hybridized to metaphase chromosomal spreads, then detected with anti-DIG-alkaline phosphate conjugate and the HNPP/Fast Red detection reaction as in Figure 1. The HNPP/Fast Red signal was viewed under a filter that was optimal for DAPI (emission maximum, 470 nm), but not for HNPP (emission maximum, 562 nm). The fluorescence was photographed directly without digital manipulation. Chromosomes were counterstained with DAPI.

109

5

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to cells

Combined DNA in situ hybridization and immunocytochemistry for the simultaneous detection of nucleic acid sequences, proteins, and incorporated BrdU in cell preparations E. J. M . Speel, F. C . S. R am aek ers, and A . H . N . H opm an, D epartm ent of M olecular C ell Biology & G enetics, U niv ersity of L im burg, M aastricht, T he N etherlands.

The combination of in situ hybridization (ISH ) and immunocytochemistry (IC C ) enables us, e.g., to simultaneously demonstrate mRN A and its protein product in the same cell, to immunophenotype cells containing a specific chromosomal aberration or viral infection, or to characterize cytokinetic parameters of tumor cell populations that are genetically or phenotypically aberrant. The factors that determine the success and sensitivity of a combination IC C and nonradioactive ISH procedure include:

Fluorochrom es have been used mainly on acetone-fixed cell preparations, since the material can be mildly post-fixed (usually with paraformaldehyde) after antigen detection and used directly for fluorescence ISH without any further pretreatment (Weber-Matthiesen et al., 1993). H owever, amplification steps for both IC C and ISH signals are often necessary for clear visualization. In such cases, enzymatic ISH pre-treatment after IC C lowers fluorescent IC C staining dramatically (Speel et al., 1994a).

! Preservation of cell morphology and

Enz ym e precipitation reactions have also been used efficiently for combined IC C / ISH staining of proteins in cell preparations and tissue sections. Enzyme precipitation products that withstand the proteolytic digestion and denaturation steps used in the ISH procedure include:

protein epitopes ! Accessibility of nucleic acid targets ! Lack of cross-reaction between the different detection procedures ! G ood color contrast ! Stability of enzyme cytochemical precipitates and fluorochromes

5

Since several steps in the ISH procedure (enzymatic digestion, post-fixation, denaturation at high temperatures, and hybridization in formamide) may destroy antigenic determinants, IC C usually precedes ISH in a combination procedure. A variety of such combined IC C / ISH procedures have been reported with either enzyme precipitation reactions (Mullink et al., 1989; Van den Brink et al., 1990; Knuutila et al., 1994; Speel et al., 1994b), fluorochromes (Van den Berg et al., 1991; Weber-Matthiesen et al., 1993), or a combination of both (Strehl & Ambros, 1993; Zheng et al., 1993; H erbergs et al., 1994; Speel et al., 1994a). The procedures can be subdivided into two groups, those which use fluorochromes for ICC, and those which use enzyme reactions.

110

! Several precipitates (Fast Red, N ew

Fuchsin, and BC IP/ N BT) formed by alkaline phosphatase ! The diaminobenzidine precipitate formed by horseradish peroxidase ! The BC IG (X-G al) precipitate formed by !-galactosidase In these cases, the digestion and denaturation steps of the ISH procedure remove the antibody and enzyme detection layers, but the precipitate remains firmly in place. The stability of the IC C precipitate thus prevents unwanted cross-reaction between the detection procedures for ISH and IC C . H ere we present a combined IC C / ISH procedure which describes compatible detection systems for fluorescence or brightfield microscopy (Table 1). Additionally, this procedure allows the localization of incorporated BrdU by fluorescence microscopy.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to cells

The procedures given below are modifications of previously published procedures (Speel et al., 1994a, 1994b).

Table 1: Detection systems for combined ICC/ISH1.

#

For analysis by Fluorescence microscopy Brightfield microscopy ICC 2

ISH

BrdU labeling

D etection by

Anti-antigen Ab-APase

Anti-antigen-!-gal

Visualization by APase-Fast Red

!-G al-BC IG

Probe

FITC - or AMC A-labeled nucleic acid

D IG - or biotin-labeled nucleic acid

D etection and visualization by

D irect viewing

PO - or APase-labeled antiD IG Ab or anti-biotin Ab plus PO -D AB, PO -TMB, or APase-Fast Red reaction

D etection by

AMC A- or FITC labeled 3 anti-BrdU Ab



Visualization by D irect viewing



1 Amplification steps may be necessary for detection of low amounts of antigen, nucleic acid target, or incorporated BrdU. 2 Abbreviations used: Ab, antibody; AMCA, aminomethylcoumarin acetic acid; APase, alkaline phosphatase; BCIG, bromochloroindolyl-galactoside; BrdU, bromodeoxyuridine; DAB, diaminobenzidine; DIG, digoxigenin; FITC, fluorescein isothiocyanate; !-Gal, !galactosidase; ICC, immunocytochemistry; ISH, in situ hybridization; PO, peroxidase; TMB, tetramethylbenzidine 3 The fluorochrome used in the BrdU visualization step should be different from the one used in the ISH visualization step.

I. Cell preparations and BrdU labeling " 1 (O ptional) To label cells, add BrdU

4 Incubate slides for 45 min at room "

(final concentration, 10 µM) to the culture medium 30 min before harvesting the cells (Speel et al., 1994a). 2 Prepare cultured normal diploid cells or " tumor cell lines (labeled or unlabeled) by one of the following methods: ! Slide and coverslip preparations: Grow cells on glass slides or coverslips. Fix in cold methanol (–20°C) for 5 s, then in cold acetone (4°C) for 3 x 5 s. Air dry samples and store at –20°C. N ote: A lternativ ely, use other fixativ es for the slide and cov erslip preparations.

temperature with an appropriate secondary antibody conjugate. U se the following to decide which antibody conjugate to use: ! I f you wish to detect the IC C antigen, the ISH antigen, and (optionally) BrdU labeling by fluorescence microscopy, use a secondary antibody that is conjugated to alkaline phosphatase (APase). ! I f you wish to detect the IC C antigen and the ISH antigen under brightfield microscopy, use a secondary antibody that is conjugated to !-galactosidase (!-G al). 5 Wash slides as follows: " ! 5 min with PBS containing 0.05% Tween ® 20. ! 5 min with PBS.

! C ytospins: C ytospin floating cells

onto glass slides at 1000 rpm for 5 min. Air dry samples for 1 h at room temperature. Fix and store as with slide and coverslip preparations above.

II. Detection of antigen by immunocytochemistry (ICC) " 1 Incubate slides for 10 min at room

temperature with PBS-Tween-N GS (PBS buffer containing 0.05% Tween ® 20 and 2–5% normal goat serum). 2 Incubate slides for 45 min at room tem" perature with an appropriate dilution of antigen-specific primary antibody in PBS-Tween-N G S. " 3 Wash slides for 2 x 5 min with PBS containing 0.05% Tween ® 20.

CONTENTS

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5

N ote: For am plification of the I C C signal, you m ay add a third antibody step after this w ash. For details of possible antibodies to use in an am plified three-antibody detection procedure, see Table 1 of the article “M ultiple target D N A in situ hybridiz ation w ith enz ym e-based cytochem ical detection system s” on page 102 of this m anual. 6 Visualize the antibody-antigen com"

plexes according to either Procedure IIIA (for APase conjugates) or Procedure IIIB (for !-G al conjugates).

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to cells

III. Visualization of ICC antigen IIIA. APase-Fast Red reaction (for producing a red precipitate visible under either fluorescence or brightfield microscopy) " 1 Mix color reagent just before use: ! 4 ml TM buffer [200 mM Tris-H C l

(pH 8.5), 10 mM MgC l2] containing 5% polyvinyl alcohol (PVA, MW 40,000; Sigma). ! 250 µl TM buffer containing 1 mg naphthol-ASMX-phosphate (Sigma). ! 750 µl TM buffer containing 5 mg Fast Red TR salt (Sigma). 2 O verlay each sample with 100 µl color " reagent and a coverslip. " 3 Incubate samples for 5–15 min at 37°C . C aution: M onitor the enz ym e reaction under a m icroscope and adjust the reaction tim e to k eep the precipitate from becom ing so dense that it shields nucleic acid sequences from the I SH detection step. 4 Wash samples 3 x 5 min with PBS. "

N ote: D o not dehydrate sam ples after w ashing. IIIB. !-Gal-BCIG reaction (for producing a blue precipitate visible under brightfield microscopy) " 1 Mix color reagent: ! 2.5 µl 5-bromo-4-chloro-3-indolyl-

5

!-D -galactoside (BC IG ; X-G al) stock solution [20 mg/ ml BC IG (X-G al) in N ,N -dimethylformamide]. ! 100 µl diluent (PBS containing 0.9 mM MgC l2, 3 mM potassium ferricyanide, and 3 mM potassium ferrocyanide). 2 O verlay each sample with 100 µl color " reagent and a coverslip. " 3 Incubate samples for 15–60 min at 37°C . C aution: M onitor the enz ym e reaction under a m icroscope and adjust the reaction tim e to k eep the precipitate from becom ing so dense that it shields nucleic acid sequences from the I SH detection step. 4 Wash samples 3 x 5 min with PBS. "

IV. Cell processing for in situ hybridization " 1 Wash slides for 2 min at 37°C with

10 mM H C l.

2 D igest samples with pepsin as follows: " ! O verlay each sample with pepsin

solution (100 µg/ ml pepsin in 10 mM H C l). ! Incubate cell samples for 10–20 min at 37°C . " 3 Wash samples for 2 min at 37°C with 10 mM H C l. N ote: For cells stained w ith the !-G alBC I G reaction, you m ay (if you w ish) dehydrate the slides after w ashing them . 4 Post-fix samples for 20 min at 4°C with "

PBS containing 1% paraformaldehyde.

5 Wash samples as follows: " ! 5 min with PBS. ! 5 min with 2 x SSC .

V. In situ hybridization (ISH) " 1 Perform a standard ISH detection and

visualization procedure on the slides as described elsewhere in this manual. U se either: ! Fluorescence-based procedures (FITC, AMC A) (when APase-conjugated antibodies were used for IC C ). ! Enzyme-based procedures (PO -D AB, PO -TMB, APase-Fast Red) (when !-G al-conjugated antibodies were used for IC C ). Example: For a detailed description of enz ym e-based I SH procedures, see “M ultiple target D N A in situ hybridiz ation w ith enz ym e-based cytochem ical detection system s” on page 100 of this m anual. 2 Include appropriate controls in the ISH "

procedure to ensure the lack of crossreaction between IC C and ISH . N ote:T he I C C precipitate rem ains firm ly in place during I SH . T he stability of the I C C precipitate usually prev ents unw anted cross-reaction betw een the detection procedures for I SH and I C C .

N ote:I f you w ish, dehydrate the sam ples after w ashing.

112

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to cells

VI. Fluorescence detection of BrdU (optional) " 1 After performing the APase-Fast Red

IC C (Procedure IIIA) and fluorescence ISH (Procedure V) reactions on BrdU labeled cells, incubate the cells for 45 min at room temperature with a specific antiBrdU antibody. 2 Wash slides for 2 x 5 min with PBS " containing 0.05% Tween ® 20. " 3 Incubate samples with a secondary antibody that is conjugated to a fluorochrome not used in the IC C or ISH reactions. Example: U se an alk aline-phosphatase conjugated antibody and the A PaseFast R ed for I C C ; a FI T C -conjugated antibody for I SH ; and an A M C A conjugated antibody for BrdU detection. N ote: I f necessary, use an am plified three-antibody detection procedure as outlined in the article “M ultiple target D N A in situ hybridiz ation w ith enz ym ebased cytochem ical detection system s” on page 100 of this m anual.

For brightfield microscopy (for detection of !-Gal-BCIG ICC and enzyme-based ISH ): D epending on the enzyme reactions used, embed specimens in one of the following: ! Aqueous, organic, or protein embedding medium, as described in the article “Multiple target D N A in situ hybridization with enzyme-based cytochemical detection systems” on page 100 of this manual. ! Entellan (Merck) organic embedding medium and immersion oil (Zeiss) (for peroxidase-D AB or peroxidase-TMB reactions only). ! Tris-glycerol mixture [1:9 (v/ v) mix of 200 mM Tris-H C l (pH 7.6) and glycerol] (for peroxidase-D AB or phosphataseFast Red reactions only).

Results $ Figure 1: Combined ICC and fluorescence

4 Before embedding, wash slides as follows: " ! 2 x 5 min with PBS containing 0.05%

ISH on lung tumor cell line EPLC65, showing cytokeratin filaments, chromosome 1, and chromosome 17. The cytokeratins (red) were visualized with a monoclonal anti-cytokeratin antibody (Ab), an alkaline phosphatase-conjugated goat anti-mouse Ab, and the APase-Fast Red reaction. The centromere of chromosome 1 (blue) was visualized with a biotinylated probe, avidinAMCA, a biotinylated goat anti-avidin Ab, and avidin-AMCA. The centromere of chromosome 17 (green) was visualized directly with a FITC-labeled probe.

Tween ® 20.

! 5 min with PBS.

5 Include appropriate controls to ensure "

that the BrdU detection step does not cross-react with ISH detection.

VII. Embedding For fluorescence microscopy (for detection of APase-Fast Red ICC, fluorescence ISH , and BrdU ): Embed specimens in a Tris-glycerol mixture [1:9 (v/ v) mix of 0.2 M Tris-H C l (pH 7.6) and glycerol] containing 2% D ABC O (Sigma) and (optionally) 0.5 µg/ ml blue D API (Sigma).

$ Figure 2: Combined ICC and fluo-

a

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b

rescence ISH on EPLC 65 cells, showing the nuclear proliferation marker Ki67, chromosome 7, and incorporated BrdU. Panel a: The Ki67 antigen (red) was visualized with a rabbit anti-Ki67 Ab, alkaline phosphatase-conjugated swine antirabbit Ab, and the APase-Fast Red reaction. The centromere of chromosome 7 (green) was visualized directly with a FITClabeled probe. Panel b: Incorporated BrdU (blue) was visualized with monoclonal anti-BrdU Ab, biotinylated horse anti-mouse Ab, and avidin-AMCA.

113

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to cells

%

Figure 3: Combined ICC and enzyme-based ISH on a human umbilical vein endothelial cell, showing the intermediate filament protein vimentin, chromosome 1, and chromosome 7. Vimentin (blue) was visualized with monoclonal anti-vimentin Ab, !-galactosidase-conjugated goat anti-mouse Ab, and the !-galactosidase-BCIG reaction. The centromeres of chromosome 1 (biotinylated probe, brown) and chromosome 7 (digoxigenin-labeled probe, red) were visualized with avidin-peroxidase, rabbit antidigoxigenin Ab and alkaline phosphatase-conjugated swine anti-rabbit Ab, the APase-Fast Red reaction, and the PO-DAB reaction. Samples were embedded in a Tris-glycerol mixture, but were not counterstained. Slides were viewed by brightfield microscopy.

References Herbergs, J.; De Bruine, A. P.; Marx, P. T. J.; Vallinga, M. I. J.; Stockbrügger, R. W.; Ramaekers, F. C. S.; Arends, J. W.; Hopman, A. H. N. (1994) Chromosome aberrations in adenomas of the colon. Proof of trisomy 7 in tumor cells by combined interphase cytogenetics and immunocytochemistry. Int. J. Cancer 57, 781–785. Knuutila, S.; Nylund, S. J.; Wessman, M.; Larramendy, M. L. (1994) Analysis of genotype and phenotype on the same interphase or mitotic cell. A manual of MAC (morphology antibody chromosomes) methodology. Cancer Genet. Cytogenet. 72, 1–15.

5

Mullink, H.; Walboomers, J. M. M.; Tadema, T. M.; Jansen, D. J.; Meijer, C. J. L. M. (1989) Combined immuno- and non-radioactive hybridocytochemistry on cells and tissue sections: influence of fixation, enzyme pre-treatment, and choice of chromogen on detection of antigen and DNA sequences. J. Histochem. Cytochem. 37, 603–609. Speel, E. J. M.; Herbergs, J.; Ramaekers, F. C. S.; Hopman, A. H. N. (1994a) Combined immunocytochemistry and fluorescence in situ hybridization for simultaneous tricolor detection of cell cycle, genomic, and phenotypic parameters of tumor cells. J. Histochem. Cytochem. 42, 961–966.

Strehl, S.; Ambros, P. F. (1993) Fluorescence in situ hybridization combined with immunohistochemistry for highly sensitive detection of chromosome 1 aberrations in neuroblastoma. Cytogenet. Cell Genet. 63, 24–28. Van Den Berg, H.; Vossen, J. M.; Van Den Bergh, R. L.; Bayer, J.; Van Tol, M. J. D. (1991) Detection of Y chromosome by in situ hybridization in combination with membrane antigens by two-color immunofluorescence. Lab. Invest. 64, 623–628. Van Den Brink, W.; Van Der Loos, C.; Volkers, H.; Lauwen, R.; Van Den Berg, F.; Houthoff, H.; Das, P. K. (1990) Combined !-galactosidase and immunogold/ silver staining for immunohistochemistry and DNA in situ hybridization. J. Histochem. Cytochem. 38, 325–329. Weber-Matthiesen, K.; Deerberg, J.; Müller-Hermelink, A.; Schlegelberger, B.; Grote, W. (1993) Rapid immunophenotypic characterization of chromosomally aberrant cells by the new fiction method. Cytogenet. Cell. Genet. 63, 123–125. Zheng, Y.-L.; Carter, N. P.; Price, C. M.; Colman, S. M.; Milton, P. J.; Hackett, G. A.; Greaves, M. F.; FergusonSmith, M. A. (1993) Prenatal diagnosis from maternal blood: simultaneous immunophenotyping and FISH of fetal nucleated erythrocytes isolated by negative magnetic cell sorting. J. Med. Genet. 30, 1051–1056.

Speel, E. J. M.; Jansen, M. P. H. M.; Ramaekers, F. C. S.; Hopman, A. H. N. (1994b) Anovel triple-color detection procedure for brightfield microscopy, combining in situ hybridization with immunocytochemistry. J. Histochem. Cytochem. 42, 1299–1307. Speel, E. J. M.; Ramaekers, F. C. S.; Hopman, A. H. N. (1995) Detection systems for in situ hybridization, and the combination with immunocytochemistry. Histochem. J., in press.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to cells

Reagents available from Boehringer Mannheim for this procedure Reagent

Description

Cat. No.

Pack size

5-Bromo-2'-deoxyuridine

One tablet contains approx. 50 mg 5-bromo-2'-deoxy-uridine and approx. 20 mg binder.

586 064

50 tablets

Pepsin

Aspartic endopeptidase with broad specificity

108 057 1 693 387

1g 5g

Digoxigenin-11-dUTP, alkali-stable

Tetralithium salt, 1 mM solution

1 093 088

25 nmol (25 µl) 125 nmol (125 µl) 5x125 nmol (5x125 µl)

1 558 706 1 570 013 Fluorescein-12-dUTP

Tetralithium salt, 1 mM solution

1 373 242

25 nmol (25 µl)

Tetramethylrhodamine-6-dUTP

Tetralithium salt, 1 mM solution

1 534 378

25 nmol (25 µl)

AMCA-6-dUTP

Tetralithium salt, 1 mM solution

1 534 386

25 nmol (25 µl)

Biotin-16-dUTP

Tetralithium salt, 1 mM solution

1 093 070

50 nmol (50 µl)

DNase I

Lyophilizate

104 159

100 mg

DNA Polymerase I

Nick Translation Grade

104 485 104 493

500 units 1000 units

Anti-Digoxigenin-AP

Fab Fragments from sheep

1 093 274

150 U (200 µl)

5

CONTENTS

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115

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to cells

In situ hybridization to mRNA in in vitro cultured cells with DNA probes D epartm ent of C ytochem istry and C ytom etry, U niv ersity of L eiden, T he N etherlands.

The protocol given below has been developed with a cell line (rat 9G ) which has integrated into its genome the major immediate early transcription unit 1 of human cytomegalovirus (Boom et al., 1986). The protocol is written in general terms and is applicable to abundantly expressed mRN A species. More information concerning this specific in situ hybridization application can be found in Raap et al. (1991).

I. Cell preparation, fixation and permeabilization N ote: A ll solutions m ust be treated w ith R N ase inhibitors. ! 1 C ulture cells at 37° C in a 5% C O 2

atmosphere on poly-L-lysine coated microscopic slides. As culture medium, use Dulbecco’s minimal essential medium without phenol red. 2 Wash the cells with PBS at 37°C , then ! fix at room temperature for 30 min in a solution of 4% (w/ v) formaldehyde, 5% (v/ v) acetic acid, and 0.9% (w/ v) N aC l. ! 3 Wash the fixed cells with PBS at room temperature and store them in 70% ethanol at 4°C . 4 Before in situ hybridization, treat the ! fixed cells as follows: " D ehydrate by incubating successively in 70% , 90% , and 100% ethanol. " Wash in 100% xylene to remove residual lipids. " Rehydrate by incubating successively in 100% , 90% , and 70% ethanol. " Finally, incubate in PBS. 5 Treat the fixed cells at 37°C with 0.1% ! (w/v) pepsin in 0.1 N H Cl, to increase permeability to macromolecular reagents. 6 Finally, treat the fixed cells as follows: ! " Wash with PBS for 5 min. " Post-fix with 1% formaldehyde for 10 min. " Wash again with PBS.

5

116

II. In situ hybridization ! 1 Label a suitable probe D N A with dig-

oxigenin (D IG ), using any of the protocols given elsewhere in this manual. 2 Prepare hybridization solution which ! contains 60% deionized formamide, 300 mM N aC l, 30 mM sodium citrate, 10 mM ED TA, 25 mM N aH 2PO 4 (pH 7.4), 5% dextran sulfate, and 250 ng/ µl sheared salmon sperm D N A. ! 3 D enature the D IG -labeled probe D N A at 80° C shortly before use and add it to the hybridization solution at a concentration of 5 ng/ µl. 4 Add 10 µl of the hybridization mixture ! (hybridization solution plus denatured probe) to the fixed, permeabilized cells and cover with an 18 x 18 mm coverslip. N ote: A n in situ denaturation step is optional. I nclusion of such a step m ay intensify the R N A signal since it m ak es the sam ple m ore accessible to the probe. 5 H ybridize at 37°C for 16 h. !

III. Washes ! 1 After hybridization, remove coverslips

by shaking the slides at room temperature in a solution of 60% formamide, 300 mM N aC l, and 30 mM sodium citrate . 2 U sing the same formamide-salt solution ! as in Step 1, wash the slides as follows: " Wash 3 x at room temperature. " Wash 1x at 37°C . ! 3 Finally, wash the slides 1 x 5 min in PBS.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to cells

! 7 Embed the cell samples in an anti-fading

IV. Immunofluorescent detection

solution which contains: " 9 parts glycerol plus " 1 part staining mixture: 1 M TrisH C l (pH 7.5), 2% 1,4-diaza-bicyclo[2,2,2]-octane (D ABC O ), and a D N A counterstain [either propidium iodide (500 ng/ ml) or D API (75 ng/ µl)].

N ote: O ther protocols for am plifying the signal m ay also be used. See the protocols under “Single color fluorescent detection w ith im m unological am plification” on page 62. ! 1 Block

non-specific binding by the following steps: " Add 100 µl blocking solution [100 mM Tris-H C l; pH 7.5, 150 mM N aC l, 0.5% (w/ v) blocking reagent] to each slide. " C over with a 24 x 50 mm coverslip. " Place slide in a moist chamber. 2 To loosen the coverslips, wash the slides ! briefly with the buffer used for immunological detection. ! 3 D etect the hybridized D IG -labeled probe by incubating the slides in a moist chamber for 45 min with a 1:500 dilution of anti-D IG -fluorescein in blocking solution (from Step 1) . 4 Wash slides with a solution of 100 mM ! Tris-H C l, pH 7.5; 150 mM N aC l; 0.05% Tween ® 20. 5 To dehydrate the cell samples, incubate ! the slides for 5 min in each of a series of ethanol solutions: 70% , 90% , then 100% ethanol. 6 Air dry the slides. ! Figure 1: Nuclear staining of integrated IE viral DNA after induction with cycloheximide. The signal throughout the cytoplasm represents viral mRNA. The two panels show the same signal with different counterstains.

Results

Panel a: PI counterstain.

5

"

Panel b: DAPI counterstain.

References Boom, R.; Geelen, J. L.; Sol, C. J.; Raap, A. K.; Minnaar, R. P.; Klaver, B. P.; Noordaa, J. v. d. (1986) Establishment of a rat cell line inducible for the expression of human cytomegalovirus immediate – early gene products by protein synthesis inhibition. J. Virol. 58, 851–859. Raap, A. K.; Van de Rijke, F .M.; Dirks, R. W.; Sol, C. J.; Boom, R.; Van der Ploeg, M. (1991) Bicolor fluorescence in situ hybridization to intron- and exon mRNA sequences. Exp. Cell Res. 197, 319–322.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to cells

Reagents available from Boehringer Mannheim for this procedure Reagent

Description

Pepsin

Aspartic endopeptidase with broad specificity

Tween® 20

Cat. No.

Pack size

108 057 1 693 387

1g 5g

1 332 465

5x10 ml

Blocking Reagent

Powder

1 096 176

50 g

Anti-Digoxigenin Fluorescein

Fab Fragments from sheep

1 207 741

200 µg

DAPI

Fluorescence dye for staining of chromosomes

236 276

10 mg

Digoxigenin-11-dUTP, alkali-stable

Tetralithium salt, 1 mM solution

1 093 088

25 nmol (25 µl) 125 nmol (125 µl) 5x125 nmol (5x125 µl)

1 558 706 1 570 013

5

Fluorescein-12-dUTP

Tetralithium salt, 1 mM solution

1 373 242

25 nmol (25 µl)

Tetramethylrhodamine-6-dUTP

Tetralithium salt, 1 mM solution

1 534 378

25 nmol (25 µl)

AMCA-6-dUTP

Tetralithium salt, 1 mM solution

1 534 386

25 nmol (25 µl)

Biotin-16-dUTP

Tetralithium salt, 1 mM solution

1 093 070

50 nmol (50 µl)

DNase I

Lyophilizate

104 159

100 mg

DNA Polymerase I

Nick Translation Grade

104 485 104 493

500 units 1000 units

808 261 808 270 808 288

250 g 500 g 1 kg

EDTA

118

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to cells

Identification of single bacterial cells using DIG-labeled oligonucleotides B. Z arda, D r. R . A m ann, and Prof. D r. K.-H . Schleifer, I nstitute for M icrobiology, Technical U niv ersity of M unich, G erm any. N ote: A n earlier v ersion of this procedure has been published (Z arda et al., 1991).

I. Organisms and growth conditions ! 1 To prepare cells needed for this experi-

ment, do the following: " Allow Escherichia coli (D eutsche Sammlung von Mikroorganismen und Zellkulturen G mbH , D SM 30083) to grow aerobically in YT broth (tryptone, 10 g/L; yeast extract, 5 g/L; glucose, 5 g/L; sodium chloride, 8 g/L; pH 7.2) at 37°C. " C ultivate Pseudom onas cepacia (D SM 50181) aerobically in M1 broth (peptone, 5 g/ L; malt extract, 3 g/ L; pH 7.0) at 30°C . Note: Cells from Methanococcus vannielii (D SM 1224) w ere a generous gift of D r. R . H uber (D ept. of M icrobiology, U niv ersity of R egensburg, FR G ). 2 To guarantee a high cellular rRN A !

content, harvest all cells at midlogarithmic phase by centrifugation in a microcentrifuge (5000 x g, 1 min). ! 3 D iscard the growth medium from the cell pellet and resuspend cells thoroughly in phosphate buffered saline (130 mM sodium chloride, 10 mM sodium phosphate; pH 7.2).

II. Cell fixation and preparation of cell smears N ote: T his procedure w as adapted from A m ann et al., 1990. ! 1 Fix the cells by adding 3 volumes of para-

formaldehyde solution (4% paraformaldehyde in PBS) to 1volume of suspended cells. 2 After 3 h incubation, do the following: ! " Pellet cells by centrifugation. " Remove the supernatant. " Wash the cell pellet with PBS. " Resuspending the cells in an aliquot of PBS. N ote: A fter adding 1 v olum e ethanol to the resuspended cells, you m ay store the cell suspension at –20°C for up to 3 m onths w ithout apparent influence on the hybridiz ation results. ! 3 Spot these fixed cell suspensions onto

5

thoroughly cleaned glass slides and allow to air dry for at least 2 h. 4 D ehydrate the cell samples by immers! ing the slides successively in solutions of 50% ethanol, then 80% ethanol, then 98% ethanol (3 min for each solution).

III. DIGlabeling of oligonucleotides with DIG-ddUTP Label oligonucleotides either according to the protocols in C hapter 4 of this manual or at the 5' end according to the protocol of the D IG O ligonucleotide 5'-End Labeling Set*.

*Sold under the tradename of Genius in the US.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to cells

IV. In situ hybridization using digoxigenin-labeled oligonucleotides ! 1 Prepare hybridization solution (900 mM

sodium chloride, 20 mM Tris-H C l, 0.01% sodium dodecyl sulfate; pH 7.2). 2 D epending on the method of analysis, ! do either of the following: " I f you will use fluorescently-labeled antibodies (Procedure Va below), proceed directly to Step 3. " If you will use peroxidase-conjugated antibodies (Procedure Vb below), then: " Prior to hybridization, incubate fixed cells for 10 min at 0°C with 1 mg/ ml of lysozyme in TE (100 mM TrisH C l, 50 mM ED TA; pH 8.0). " Remove lysozyme by thoroughly rinsing the slide with sterile H 2O . " Air dry the slide. " Proceed to Step 3. ! 3 Add 8 µl hybridization solution containing 50 ng of labeled oligonucleotide probe to the prepared slide (from Procedure II). 4 Incubate the slide for 2 h at 45°C in an ! isotonically equilibrated humid chamber. Va. Detection of DIG-labeled oligonucleotides with fluorescently labeled anti-DIG Fab fragments ! 1 D ilute fluorescein- or rhodamine-labeled

5

anti-D IG Fab fragments 1:4 in blocking solution (150 mM sodium chloride; 100 mM Tris-H C l, pH 7.5; 0.5% bovine serum albumin; and 0.5% blocking reagent). 2 Add 10 µl of the diluted antibody to the ! slide and incubate the slide for another hour at 27°C in the humid chamber. ! 3 Remove the slide from the humid chamber and immerse in 40 ml of a washing solution (150 mM sodium chloride, 100 mM Tris-H Cl, 0.01% SDS; pH 7.4) at 29°C for 10 min. 4 Prepare the slides for viewing by doing ! the following: " Rinse the slides briefly with sterile water (which has been filtered through a 0.2 µm filter). " Air dry. " Mount in C itifluor (C itifluor Ltd., London, U .K.).

120

5 View the cell smears with a Plan!

N eofluar 100 x objective (oil immersion) on the following setup: a Zeiss Axioplan microscope fitted for epifluorescence microscopy with a 50 W mercury high pressure bulb and Zeiss filter sets 09 and 15 (Zeiss, O berkochen, FRG ). 6 For photomicrographs, use Kodak Ekta! chrome P1600 color reversal film and an exposure time of 15 s for epifluorescence or 0.01 s for phase contrast micrographs. Vb. Detection of DIG-labeled oligonucleotides with peroxidaseconjugated anti-DIG Fab fragments ! 1 U se the same conditions for hybridiza-

tion and antibody binding as for fluorescent antibodies (Procedures IV and Va), except include a lysozyme pretreatment of the cell smears prior to hybridization. For details, see Procedure IV above. 2 Visualize the bound antibody as follows: ! " Prepare peroxidase substrate-nickle chloride solution [1.3 mM diaminobenzidine, 0.02% (v/ v) H 2O 2, 0.03% (w/ v) nickel chloride, 5 mM Tris-H C l (pH 7.4)]. " Incubate slide with peroxidase substrate-nickle chloride solution until a purplish-blue precipitate forms. ! 3 View slides under a light microscope and photograph with Kodak Ektachrome P1600 color reversal film.

Results U sing the techniques in this article, we could detect P. cepacia cells in a mixed sample of three bacteria (Figure 1). Additional experiments with a D IG -labeled oligonucleotide not complementary to rRN A show no significant levels of nonspecifically bound D IG -labeled nucleic acid probes and anti-D IG antibodies.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to cells

# Figure 1: Specific identification of whole fixed cells of P. cepacia in a mixture of P. cepacia (chains of rods), E. coli (rods) and M. van-nielii (cocci) with a DIG-

a

labeled oligonucleotide probe. Phase contrast (panel a) and epifluorescence (panel b) photos show detection with fluorescently labeled anti-DIG Fab fragments (Zarda et al., 1991). Phase contrast (panel c) and brightfield (panel d) photos show detection with peroxidase-conjugated anti-DIG Fab fragments

b

c

Reprinted from Zarda, B.; Amann, R.; Wallner, G.; Schleifer, K. H. (1991) Identification of single bacterial cells using digoxigenin-labeled rRNA-targeted oligonucleotides. J. Gen. Microbiol. 137, 2823–2830 with permission of the society for general microbiology.

d

References Amann, R. I.; Binder, B. J.; Olson, R. J.; Chisholm, S. W.; Devereux, R.; Stahl, D. A. (1990) Combination of 16 S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl. Environ. Microbiol. 56, 1919–1925. Mühlegger, K.; Huber, E.; von der Eltz, H.; Rüger, R.; Kessler, C. (1990) Nonradioactive labeling and detection of nucleic acids. Biol. Chem. Hoppe-Seyler 371, 953–965. Reagent

Description

DIG-Oligonucleotide 3'-End Labeling Kit*

Zarda, B.; Amann, R.; Wallner, G.; Schleifer, K. H. (1991) Identification of single bacterial cells using digoxigenin-labeled rRNA-targeted oligonucleotides. J. Gen. Microbiol. 137, 2823–2830.

Reagents available from Boehringer Mannheim for this procedure Cat. No.

Pack size

3'-end labeling of oligonucleotides from 14 to 100 nucleotides in length with DIG-11-ddUTP and terminal transferase

1 362 372

1 Kit (25 labeling reactions)

DIG-Oligonucleotide 5'-End Labeling Set*

With the DIGOligonucleotide 5'-End Labeling Set, oligonucleotides can be labeled with digoxigenin at the 5’-end after synthesis that includes the addition of a phosphoramidite

1 480 863

1 Set (10 labeling reactions)

Anti-DigoxigeninFluorescein

Fab Fragments from sheep

1 207 741

200 µg

Anti-DigoxigeninRhodamine

Fab Fragments from sheep

1 207 750

200 µg

Anti-Digoxigenin-POD

Fab Fragments from sheep

1 207 733

150 U

Tris

Powder

127 434 708 968 708 976

100 g 500 g 1 kg

EDTA

Powder

808 261 808 270 808 288

250 g 500 g 1 kg

Blocking Reagent

Powder

1 096 176

50 g

BSA

Highest quality lyophilizate

238 031 238 040

1g 10 g

SDS

Purify 99%

1 028 685 1 028 693

100 g 500 g

CONTENTS

INDEX

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*Sold under the tradename of Genius in the US.

121

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to Tissues

Detection of HPV 11 DNA in paraffin-embedded laryngeal tissue with a DIG-labeled DNA probe D r. J. R olighed and D r. H . L indeberg, EN T-departm ent and I nstitute for Pathology, A arhus U niv ersity H ospital, D enm ark .

The technique of tissue in situ hybridization has become a powerful tool in pathology, although it is still mainly used for research purposes. The most important application at the moment is probably the demonstration of viral DN A (or RN A) in biopsies. This technique has made it possible to demonstrate infection with CMV (Grody et al., 1987; Unger et al., 1988), H BV (Blum et al., 1983; N egro et al., 1985), parvovirus (Proter et al., 1988), coxsackie B (Archard et al., 1987) EBV and H IV (Pezzella et al., 1989). In situ hybridization has been widely used to demonstrate H PV sequences in biopsies from the uterine cervix as well as from the head and neck (Lindeberg et al., 1990; Lindeberg et al., 1989; Shah et al., 1988). Although some of these viruses can be demonstrated by other methods, in situ hybridization is much faster than methods using infection of tissue cultures; for H PV tissue culture methods do not exist. Radioactively labeled probes were used originally, but in recent years nonradioactive probes have become increasingly popular for obvious reasons.

5

In general, the use of radioactively labeled probes is considered superior to other methods. H owever, this is probably not correct (N uevo and Richart, 1989). For instance, H PV type 16 was demonstrated in SiH a cells by in situ hybridization with digoxigenin-labeled probes (H eiles et al., 1988). As SiH a-cells contain only 1–2 H PV copies per cell, one can hardly ask for a further improvement in sensitivity.

*Sold under the tradename of Genius in the US.

122

There is an increasing demand from histopathologists for simple in situ hybridization methods, so that laboratory technicians can perform, for example, typing of H PV as a daily routine. To answer this demand, we present here a protocol for the detection of H PV DN A type 11 in laryngeal papillomas as well as in genital condylomatous lesions. The method is used on routine material which has been fixed in formalin and embedded in paraffin. The method described has the following advantages: ! The method is fast; results are obtained in about 5–6 h, and the hands-on time is about 2 h.

! The method is economical; the DIG

Labeling and Detection Kit* is sufficient for preparing 10 ml of DIG-labeled probe cocktail (1 µl labeled DN A/ml). This is enough for hybridizing 1000 sections. ! Problems linked to endogenous biotin are avoided, since endogenous digoxigenin apparently does not exist. The main steps in performing in situ hybridization on formalin-fixed, paraffinembedded specimens are: " 1 Exposure of target DN A. This is done with a carefully controlled proteolytic digestion step. 2 Denaturation of ds DN A, followed by " hybridization. " 3 Washing and detection of DN A hybrids. In our experience the most difficult step is optimization of the proteolytic digestion.

I. Probe preparation " 1 Using standard methods, remove the viral

insert from its vector by restriction enzyme cleavage and separate it on a submerged agarose gel. 2 Recover the insert from the gel by " electroelution or other methods. " 3 Label the 8 kb viral insert nonradioactively with digoxigenin acccording to the procedures given in Chapter 4 of this manual. 4 To a tube, add the following ingredients " of the probe cocktail: ! 10 µl 50 x Denhart’s solution. ! 50 µl dextran sulfate 50% (w/v). ! 10 µl sonicated salmon sperm DN A (10 mg/µl). ! 100 µl 20 x SSC. ! 500 ng digoxigenin-labeled probe in 50 µl TE. ! Enough distilled water to make a total volume of 250 µl. 5 To complete the probe cocktail, add " 250 µl formamide to the tube. Tip: U se glov es w hen handling solutions containing form am ide. Form am ide should be handled in a fum e hood. 6 Mix the probe cocktail (by vortexing) and " store it at –20°C.

CONTENTS

INDEX

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to Tissues

Reagents available from Boehringer Mannheim for this procedure Reagent

Description

Cat. No.

Pack size

DIG DNA Labeling and Detection Kit* , ** , ***

Random primed labeling of DNA probes with DIG-11-dUTP, alkali-labile and color detection of DIG-labeled hybrids.

1 093 657

1 Kit (25 labeling reactions and detection of 50 blots of 100 cm2)

Proteinase K

Lyophilizate

161 519 745 723 1 000 144 1 092 766

25 mg 100 mg 500 mg 1g

Tris

Powder

127 434 708 968 708 976

100 g 500 g 1 kg

EDTA

Powder

808 261 808 270 808 288

250 g 500 g 1 kg

Blocking Reagent

Powder

1 096 176

50 g

Anti-DIG-AP

Fab Fragments from sheep

1 093 274

150 U (200 µl)

NBT/BCIP

Stock solution

1 681 451

8 ml

***Sold under the tradename of Genius in the US. ***EP Patent 0 324 474 granted to Boehringer Mannheim GmbH. ***Licensed by Institute Pasteur.

References Archard, L. C.; Bowles, N. E.; Olsen, E. G. J.; Richardson, P. J. (1987) Detection of persistent coxsachie B virus RNA in dilated cardiomyopathy and myocarditis. Eur. Heart J. (Suppl.) 8, 437–440.

Nuevo, G. J.; Richart, R. M. (1989) A comparison of Biotin and 35S-based in situ hybridization methodologies for detection of human papillomavirus DNA. Lab. Invest. 61, 471–475.

Blum, H. E.; Stowringer, L.; Figue, A. et al. (1983) Detection of Hepatitis B virus DNA in hepatocytes, bile duct epithelium, and vascular elements by in situ hybridization. Proc. Natl. Acad. Sci. (USA) 80, 6685–6688.

Pezzella, M.; Pezzella, F.; Rapicetta, M. et al. (1989) HBV and HIV expression in lymph nodes of HIV positive LAS patients: histology and in situ hybridization. Mol. Cell Probes 3, 125–132.

Grody, W. W.; Cheng, L.; Lewin, K. J. (1987) In situ viral DNA hybridization in diagnostic surgical pathology. Hum. Pathol. 18, 535–543. Heiles, H. B. J.; Genersch, E.; Kessler, C.; Neumann, R.; Eggers, H. J. (1988) In situ hybridization with digoxigenin-labeled DNA of human papillomaviruses (HPV 16/18) in HeLa and SiHa cells. BioTechniques 6 (10), 978–981. Lindeberg, H.; Johansen, L. (1990) The presence of human papillomavirus (HPV) in solitary adult laryngeal papillomas demonstrated by in situ DNA hybridization with sulphonated probes. Clin. Otolaryngol. 15, 367–371.

5

Proter, H. J.; Quantrill, A. M.; Fleming, K. A. (1988) B19 parvovirus infection of myocardial cells. Lancet 1, 535–536. Shah, K. V.; Gupta, J. W.; Stoler, M. H. (1988) The in situ hybridization test in the diagnosis of human papillomaviruses. In: De Palo, G.; Rilke, F.; zur Hausen, H. (Eds.) Serono Symposia 46: Herpes and Papillomaviruses. New York: Raven Press, 151–159. Unger, E. R.; Budgeon, L. R.; Myorson, B.; Brigati, D. J. (1988) Viral diagnosis by in situ hybridization. Description of a rapid simplified colorimetric method. Am J. Surg. Pathol. 18, 1–8.

Lindeberg, J.; Syrjänen, S.; Karja, J.; Syrjänen, K. (1989) Human papillomavirus type 11 DNA in squamous cell carcinomas and preexisting multiple laryngeal papillomas. Acta Oto-Laryngol. 107, 141–149. Negro, F.; Berninger, M.; Chaiberge, E. et al. (1985) Detection of HBV-DNA by in situ hybridization using a biotin-labeled probe. J. Med. Virol. 15, 373–382.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to Tissues

II. Pretreatment of slides

IV. Hybridization

" 1 Wash slides overnight in a detergent, fol-

" 1 After the treatment with Proteinase K,

lowed by an intensive wash in tap water, and finally, a wash in demineralized water. 2 After drying the slides, wash them for " 3 min in acetone. " 3 Transfer the washed slides to a 2% solution of 3-aminopropyltrithoxysilane in acetone and incubate for 5 min. 4 Wash the slides briefly in distilled water " and allow to dry. N ote: T he coated slides can be stored dry under dust-free conditions for sev eral m onths.

remove the coverslips and fix the sections in 0.4% formaldehyde for 5 min at 4°C. 2 Immerse the sections in distilled water for " 5 min. " 3 Allow the sections to drip and air dry for 5 min. 4 Distribute about 5–10 µl of the probe " cocktail over each section. N ote: L arge biopsies m ay require larger v olum es of probe cock tail.

III. Preparation of tissue sections " 1 Fix biopsies from condylomatous lesions

and from laryngeal papillomas in 4% phosphate-buffered formaldehyde and embed them in paraffin. 2 Cut routine sections, float them on " demineralized water and place them on the pretreated slides. " 3 Treat the sections on the slides as follows: ! Bake the sections for 30 min at 60°C. ! Dewax in xylene. ! Rehydrate them by dipping them in a graded ethanol series (2 x in 99% ethanol, 2 x in 96% ethanol, 1x in 70% ethanol, 1x in H 2O ). 4 Immediately before use, prepare a working " Proteinase K solution as follows: ! Thaw an aliquoted frozen stock solution of Proteinase K, 10 mg/ml, in H 2O . ! Dilute the stock Proteinase K to the working concentration of 100 µg/ml in TES [50 mM Tris-H Cl (pH 7.4), 10 mM EDTA, 10 mM N aCl]. 5 Treat each section with 20–30 µl of the " working solution of Proteinase K for 15 min at 37°C. During the proteolytic digestion cover the sections with coverslips and place them in a humid chamber. Tip: We use only siliconiz ed cov erslips.

5 Prepare a negative control by covering "

one section from each block with a “blind” probe cocktail, i.e. a probe cocktail containing all the ingredients in Procedure I, except the labeled H PV DN A. 6 Place coverslips over each section and " denature the DN A by placing the slides on a heating plate at 95°C for 6 min. Tip: We use only siliconiz ed cov erslips. " 7 Cool the slides for 1 min on ice. 8 Place the slides in a humid chamber and "

incubate for 3 h at 42°C in an oven. Tip: T he hybridiz ation tim e m ay be prolonged ov ernight w hen conv enient.

V. Washes

5

Remove the coverslips and wash the sections as follows: ! 2 x 5 min in 2 x SSC at 20°C. ! 1 x 10 min in 0.1 x SSC at 42°C.

N ote: T he proteolytic treatm ent is crucial and m ay need to be v aried w ith different tissues.W hen too m uch Proteinase K is used, the tissue totally disintegrates, and if too little is used positiv e signals m ay be totally absent.

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VI. Detection of hybrids " 1 D ip each section into buffer 1 [100 mM

Tris-H C l, 150 mM N aC l;pH 7.5 (20°C )].

2 Cover each section with 20–40 µl buffer 2 "

[0.5% (w/v) blocking reagent in buffer 1], then add a coverslip. " 3 Incubate sections at 20°C for 15 min. 4 Remove the coverslips and dip the " sections in buffer 1. 5 Dilute the antibody conjugate 1:500 in " buffer 2. 6 Distribute 10–20 µl of the diluted con" jugate over each section, including the negative controls. " 7 Place a coverslip over each section and place all sections in a humid chamber at room temperature for 1 h. 8 Remove the coverslips, wash the sections " 2 x10 min in buffer 1, then equilibrate for 5 min in buffer 3. " 9 Distribute approx. 20 µl color-solution (N BT/BCIP) onto each section, cover with a coverslip and leave in the dark overnight. N ote: You m ay shorten the colorreaction to 30–60 m in if you w ish. I n fact, a positiv e reaction m ay be seen after a few m inutes in the dark w hen H PV is present in a high copy-num ber. Hint: U se glov es w hen handling the color solution.

5

10 After the incubation, do the following: " ! Remove the coverslips. ! Wash the sections gently. ! Stain a few seconds with neutral red. ! Mount.

C aution: A v oid dehydrating procedures since ethanol m ay rem ov e or w eak en the signal.

Results The described method is applied to biopsies of multiple and solitary laryngeal papillomas and on a flat condylomatous lesion of the uterine cervix (Figure 1). The positive results are obvious, and little background is observed in these examples. The procedure is extremely straightforward and can be performed in 5–6 h, including the 3 h hybridization. Consequently, the whole procedure can be performed in 1 day and the results recorded the next morning. If needed, the hybridization can be shortened; in our initial experiments, we were able to detect H PV type 11 after only 1 h of hybridization.

Acknowledgment Plasmids with H PV type 11 were kindly supplied by Prof. zur H ausen and co-workers, DKFZ, H eidelberg, Germany.

a

b

c

d

#

Figure 1: Detection of HPV 11 DNA in biopsies of multiple and solitary laryngeal papillomas and on a flat condylomatous lesion of the uterine cervix. Positive results are shown in Panels a and c. The negative controls (Panels b and d) show no signal.

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Detection of mRNA in tissue sections using DIG-labeled RNA and oligonucleotide probes P. Kom m inoth, D iv ision of C ell and M olecular Pathology, D epartm ent of Pathology, U niv ersity of Z ürich, Sw itz erland.

The protocols given below are modifications of recently published methods for nonisotopic in situ hybridization with digoxigenin-labeled probes (Komminoth, 1992; Komminoth et al., 1992). In those reports we demonstrated: " Digoxigenin-labeled probes are as sensi-

tive as 35S-labeled probes.

" Protocols with digoxigenin-labeled probes

may also be applied to diagnostic in situ hybridization procedures. The following protocols have been successfully used in our laboratories to detect mRN A in frozen and paraffin-embedded tissue sections with either RN A or oligonucleotide probes. RN A probes are best for high sensitivity detection procedures because: " RN A probes are easily generated and

labeled by in vitro transcription procedures. " H ybrids between mRN A and RN A probes are highly stable. " Protocols using stringent washing conditions and RN ase digestion steps yield highly specific signals with low background.

5

Synthetic oligonucleotide probes are an attractive alternative to RN A probes for the detection of abundant mRN A sequences in tissue sections, because: " Any known nucleic acid sequence can

rapidly be made by automated chemical synthesis. " Such probes are more stable than RN A probes. O ligonucleotide probes are labeled during synthesis or by addition of reporter molecules at the 5' or 3' end after synthesis. The most efficient labeling method is addition of a “tail” of labeled nucleotides to the 3' end. O ligonucleotide probes are generally less sensitive than RN A probes since fewer labeled nucleotides can be incorporated per molecule of probe.

The protocols below are written in general terms. Additional information concerning the in situ hybridization methods and important technical details are provided in Komminoth (1992), Komminoth et al. (1992), Komminoth et al. (1995), Sambrook et al. (1989), and the Boehringer Mannheim DIG/Genius™ System User’s Guide for Membrane H ybridization.

I. Preparation of slides N ote:G elatin-coated slides are excellent for large tissue sections obtained from froz en or paraffin-em bedded specim ens. A lternativ ely, silane-coated slides can be used, but are better suited for sm all tissue sam ples or cell preparations. IA. Gelatin-coated slides ! 1 Clean slides by soaking for 10 min in

Chromerge® solution (Merck).

2 Wash slides in running hot water. ! ! 3 Rinse slides in distilled water. 4 Prepare gelatin solution as follows: ! " Dissolve 10 g gelatin (Merck) in 1 liter

of distilled water which has been heated to 40°–50°C. " Add 4 ml 25% chromium potassium sulfate (CPS) solution (Merck) to the gelatin to make a final concentration of 0.1% CPS. 5 Dip slides in gelatin solution for 10 min. ! 6 Let the slides air dry. ! ! 7 Soak slides for 10 min in PBS (pH 7.4) containing 1% paraformaldehyde. 8 Let the slides air dry. ! ! 9 Bake slides at 60°C overnight. IB. Silane-coated slides ! 1 Soak clean glass slides for 60 min in silane

solution [5 ml of 3-aminopropyltriethoxysilane (Sigma) in 250 ml of acetone]. 2 Wash the slides 2 x 10 min in distilled ! water. ! 3 Dry slides overnight at 60°C. N ote: Both gelatin- and silane-coated slides can be stored for sev eral m onths under dry and dust free conditions.

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IIB. Paraffin sections

II. Tissue preparation General guidelines: Fix or freeze tissue as soon as possible after surgical excision to prevent degradation of mRN A. If possible, use cryostat sections of paraformaldehyde-fixed tissues which have been immersed in sucrose (as in Procedure IIA). These provide excellent conditions for mRN A localization and preservation of sample morphology. H owever, in surgical pathology, most tissues are routinely fixed in formalin. For mRN A localization, do not fix in formalin for more than 24 h. Prolonged storage of samples in formalin will cause covalent linkages between mRN A and proteins, making target sequences less accessible. C aution: Prepare all solutions for procedures I I A and I I B w ith distilled, deioniz ed w ater (ddH 2O ) that has been treated w ith 0.1% diethylpyrocarbonate (D EPC ) (Sam brook et al., 1989). IIA. Frozen sections ! 1 Cut tissues into 2 mm thick slices. 2 Incubate cut tissues at 4°C for 2–4 h with !

freshly made, filtered fixative (DEPCtreated PBS containing 4% paraformaldehyde; pH 7.5). ! 3 Decant fixative and soak tissues at 4°C overnight in sucrose solution [DEPCtreated PBS containing 30% sucrose (RN ase-free)]. N ote: D uring this step, the tissues should sink to the bottom of the container. 4 Store tissue samples in a freezing com!

pound at –80°C, or, for long time storage, at –140°C (N aber et al., 1992). 5 For sectioning, warm the samples to ! –20°C and cut 10 µm sections in a cryostat. 6 Place the sections on pretreated glass ! slides (from Procedure I). ! 7 Dry slides in an oven at 40°C overnight. 8 Do either of the following: ! " Use the prepared slides immediately. or " Store the slides in a box at –80°C. Before processing, warm the stored slides to room temperature and dry them in an oven at 40°C for a minimum of 2 h. N ote:Slides w ith cryostat sectionsm ay be stored at –80° C for sev eral w eek s.

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! 1 Prepare formaldehyde-fixed and paraffin-

embedded material according to standard procedures. 2 Cut 7 µm sections from the paraffin! embedded material and place the sections onto coated glass slides (from Procedure I). ! 3 Dry slides in an oven at 40°C overnight. 4 Dewax sections 2 x 10 min with fresh ! xylene. 5 Rehydrate sections in the following ! solutions: " 1 x 5 min in 100% ethanol. " 1 x 5 min in 95% ethanol. " 1 x 5 min in 70% ethanol. " 2 x in DEPC-treated ddH 2O .

IIIA. ISH protocol for detection of mRNA with DIG-labeled RNA probes C aution: Prepare all solutions for procedures below (probe labeling through posthybridiz ation) w ith distilled, deioniz ed w ater (ddH 2O ) that has been treated w ith 0.1% diethylpyrocarbonate (D EPC ) (Sam brook et al., 1989). To av oid R N ase contam ination, w ear glov es throughout the procedures and use different glassw are for pre- and posthybridiz ation steps. N ote: Perform all procedures below at room tem perature unless a different tem perature is stated.

5

Probe labeling N ote: To ensure tissue penetration, w e prefer to w ork w ith R N A probes that are ≤ 500 bases long. ! 1 Clone a cDN A insert into an RN A

expression vector (plasmid) according to standard procedures (Sambrook et al., 1989). 2 Linearize the RN A expression vector ! with appropriate restriction enzymes to allow in vitro run-off synthesis of both sense- and antisense-oriented RN A probes (Valentino et al., 1987). N ote: A s an alternativ e to lineariz ed plasm ids, use PC R -generated tem plates containing R N A polym erase prom oter sequences for in v itro transcription (Young et al., 1991).

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to Tissues

! 3 Purify linearized plasmid by phenol-

chloroform extraction and ethanol precipitation. C aution: Som e plasm id purification k its contain R N ase digestion steps for rem ov ing bacterial R N A . Traces of R N ase m ay destroy transcribed probes. To ensure rem ov al of residual R N ase, perform m ultiple phenol-chloroform extractions of the lineariz ed plasm id. 4 To avoid RN A polymerase inhibition, !

resuspend the plasmid in EDTA-free buffer or DEPC-treated ddH 2O . 5 Generate ! digoxigenin-labeled RN A probes in both the sense and antisense direction by in vitro transcription with the DIG RN A Labeling Kit* or according to procedures in Chapter 4 of this manual. 6 Purify labeled probes according to ! procedures in the DIG/Genius™ System User’s Guide for Membrane H ybridization or in Chapter 4 of this manual. C aution: Probes longer than 500 bases m ay not penetrate tissue. I f probes are longer than 500 bases, shorten them by alk aline hydrolysis according to procedures in the D I G / G enius™ System U ser’s G uide for M em brane H ybridiz ation. ! 7 Estimate the yield of labeled probes by

direct blotting procedures as described in Chapter 4 of this manual. 8 Aliquot labeled probes and store them at ! –80°C. N ote: Probes are stable for up to one year.

5

Prehybridization ! 1 Incubate sections as follows: " 2 x 5 min with DEPC-treated PBS (pH

7.4) (Sambrook et al., 1989). N ote: PBS contains 140 m M N aC l, 2.7 m M KC l, 10 m M N a2H PO 4, 1.8 m M KH 2PO 4. " 2 x 5 min with DEPC-treated PBS containing 100 mM glycine. 2 Treat sections for 15 min with DEPC! treated PBS containing 0.3% Triton ® X-100. ! 3 Wash 2 x 5 min with DEPC-treated PBS. 4 Permeabilize sections for 30 min at 37°C ! with TE buffer (100 mM Tris-H Cl, 50 mM EDTA, pH 8.0) containing either 1 µg/ml RN ase-free Proteinase K (for frozen sections) or 5–20 µg/ml RN asefree Proteinase K (for paraffin sections).

C aution: Perm eabiliz ation is the m ost critical step of the entire in situ hybridiz ation procedure. I n paraffin-em bedded archiv al m aterials, optim al tissue perm eabiliz ation differs for each case, depending upon duration and type of fixation. We recom m end titration of the Proteinase K concentration. A lternativ e perm eabiliz ation protocols for im prov ed efficiency of digestion include incubation of slides for 20–30 m in at 37°C w ith 0.1% pepsin in 0.2 M H C l. 5 Post-fix sections for 5 min at 4°C with !

DEPC-treated PBS containing 4% paraformaldehyde. 6 Wash sections 2 x 5 min with DEPC! treated PBS. ! 7 To acetylate sections, place slide containers on a rocking platform and incubate slides 2 x 5 min with 0.1 M triethanolamine (TEA) buffer, pH 8.0, containing 0.25% (v/v) acetic anhydride (Sigma). C aution: A cetic anhydride is highly unstable. A dd acetic anhydride to each change of T EA -acetic anhydride solution im m ediately before incubation. 8 Incubate sections at 37°C for at least !

10 min with prehybridization buffer [4 x SSC containing 50% (v/v) deionized formamide]. N ote: D eioniz e form am ide w ith D ow ex ® M R 3 (Sigm a) according to protocols described in Sam brook et al. (1989). N ote: 1 x SSC = 150 m M N aC l, 15 m M sodium citrate, pH 7.2. In situ hybridization ! 1 Prepare hybridization buffer containing: " 40% deionized formamide. " 10% dextran sulfate. " 1 x Denhardt’s solution [0.02% Ficoll®,

0.02% polyvinylpyrrolidone, 10 mg/ml RN ase-free bovine serum albumin]. " 4 x SSC. " 10 mM DTT. " 1 mg/ml yeast t-RN A. " 1 mg/ml denatured and sheared salmon sperm DN A. C aution: D enature and add salm on sperm D N A to buffer shortly before hybridiz ation. N ote: H ybridiz ation buffer (m inus salm on sperm D N A ) can be stored at –20° C for sev eral m onths.

*Sold under the tradename of Genius in the US.

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2 Drain prehybridization buffer from the !

! 1 Using a shaking platform, wash sections

slides and overlay each section with 30 µl of hybridization buffer containing 5–10 ng of digoxigenin-labeled RN A probe. ! 3 Cover samples with a 24 x 30 mm hydrophobic plastic coverslip (e.g. cut from Gel Bond Film, FMC BioProducts, Rockland, ME, USA). 4 Incubate sections at 42°C overnight in a ! humid chamber.

2 x 10 min with buffer 1 [100 mM TrisH Cl (pH 7.5), 150 mM N aCl]. 2 Cover sections for 30 min with blocking ! solution [buffer 1 containing 0.1% Triton ® X-100 and 2% normal sheep serum (Sigma)]. ! 3 Decant blocking solution and incubate sections for 2 h in a humid chamber with buffer 1 containing 0.1% Triton ® X-100, 1% normal sheep serum, and a suitable dilution of sheep anti-DIG-alkaline phosphatase [Fab fragments]. N ote: For optim al detection, incubate sev eral sections (from the sam e sam ple) w ith different dilutions of the antibody (1:100; 1:500, and 1:1000).

Posthybridization C aution: U se a separate set of glassw are for posthybridiz ation and prehybridiz ation procedures to av oid R N ase contam ination in prehybridiz ation steps. ! 1 Remove coverslips from sections by

immersing slides for 5–10 min in 2 x SSC. C aution: D o not place sam ples w hich are hybridiz ed w ith different probes in the sam e container. 2 In a shaking water bath at 37°C, wash !

sections as follows: " 2 x15 min with 2 x SSC. " 2 x15 min with 1x SSC. ! 3 To digest any single-stranded (unbound) RN A probe, incubate sections for 30 min at 37°C in N TE buffer [500 mM N aCl, 10 mM Tris, 1 mM EDTA, pH 8.0] containing 20 µg/ml RN ase A. C aution: Be particularly careful w ith R N ase. T his enz ym e is extrem ely stable and difficult to inactiv ate. A v oid contam ination of any equipm ent or glassw are w hich m ight be used for probe preparation or prehybridiz ation procedures. U se separate glassw are for R N ase-contam inated solutions. 4 In a shaking water bath at 37°C, wash !

sections 2 x 30 min with 0.1 x SSC. N ote: I f this procedure giv es high back ground or nonspecific signals, try posthybridiz ation w ashings at 52° C w ith 2 x SSC containing 50% form am ide. Immunological detection N ote: T his detection procedure uses com ponents of the D I G N ucleic A cid D etection Kit*. A lternativ e detection procedures include the im m unogold m ethod w ith silv er enhancem ent for enz ym e-independent probe detection (Kom m inoth et al. 1992; Kom m inoth et al., 1995) and other detection procedures described in C hapter 5 of this m anual.

4 Using a shaking platform, wash sections !

2 x10 min with buffer 1.

5 Incubate sections for 10 min with buffer 2 !

[100 mM Tris-H Cl (pH 9.5), 100 mM N aCl, 50 mM MgCl2]. 6 Prepare a color solution containing: ! " 10 ml of buffer 2. " 45 µl nitroblue tetrazolium (N BT) solution (75 mg N BT/ml in 70% dimethylformamide). " 35 µl 5-bromo-4-chloro-3-indolylphosphate (BCIP or X-phosphate) solution (50 mg X-phosphate/ml in 100% dimethylformamide). " 1 mM (2.4 mg/10 ml) levamisole (Sigma). N ote: For conv enience, prepare a 1 M stock solution of lev am isole in ddH 2O (stable for sev eral w eek s, w hen stored at 4°C ) and add 10 µl of the stock to 10 m l of the color solution. I f endogenous phosphatase activ ity is high, increase the concentration of lev am isole to 5 m M (50 µl 1 M stock / 10 m l solution) in the color solution.

5

N ote: N BT / BC I P produces a blue precipitating product. For other colors, use other alk aline phosphatase substrates. For exam ple, use either Fast R ed or I N T / BC I P for red/ brow n precipitates. ! 7 Cover each section with approximately

200 µl color solution and incubate slides in a humid chamber for 2–24 h in the dark. 8 When color development is optimal, stop ! the color reaction by incubating the slides in buffer 3 [10 mM Tris-H Cl (pH 8.1), 1 mM EDTA]. ! 9 Dip slides briefly in distilled water. *Sold under the tradename of Genius in the US.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to Tissues

10 Counterstain sections for 1–2 min with !

! 3 Add a 24 x 30 mm coverslip to each

0.02% fast green FCF or 0.1% nuclear Fast Red (Aldrich Chemical Corp., Milwaukee, WI, USA) in distilled H 2O . 11 Wash sections 2 x 10 min in tap water. ! ! 12 Mount sections using an aqueous mounting solution (e.g. Aqua-Mount from Lerner Laboratories, Pittsburgh, PA, USA). C aution: D o not use xylene-based m ounting solutions. T hese lead to crystal form ation of color precipitates.

section and incubate slides in a humid chamber at 37°C for 2 h. 4 Remove coverslips by immersing slides ! for 5 min in 2xSSC.

IIIB. ISH protocol for detecting mRNA with DIG-labeledoligonucleotideprobes

! 1 Prepare hybridization buffer containing: " 2 x SSC. " 1 x Denhardt’s solution [0.02% Ficoll®,

Probe labeling ! 1 Label 100 pmol of oligonucleotide probe

(20–30 mer) by tailing with the DIG O ligonucleotide Tailing Kit* according to the protocol described in Chapter 4 of this manual. 2 Purify labeled probes according to pro! cedures in the DIG/Genius™ System User’s Guide for Membrane H ybridization or in Chapter 4 of this manual. ! 3 Estimate the yield of labeled probes by direct blotting procedures as described in Chapter 4 of this manual. 4 Aliquot the labeled probes and store ! them at –20°C. N ote: Probes are stable for up to one year.

5

N ote: To increase the sensitiv ity of in situ hybridiz ation procedures, prepare sev eral labeled probes that are com plem entary to different regions of the target R N A , then use m ixtures of the probes for the hybridiz ation step below. Prehybridization ! 1 Follow Steps 1–7 from the prehybridi-

zation section of Procedure IIIA (for RN A probes). 2 O verlay each section with 40 µl ! prehybridization buffer (identical to the hybridization buffer to be used for in situ hybridization below, but containing no labeled probe).

In situ hybridization N ote: To increase the sensitiv ity of in situ hybridiz ation procedures, use m ixtures of oligonucleotide probes that are com plem entary to different regions of the target RN A.

0.02% polyvinylpyrrolidone, 10 mg/ml RN ase-free bovine serum albumin]. " 10% dextran sulfate. " 50 mM phosphate buffer (pH 7.0). " 50 mM DTT. " 250 µg/ml yeast t-RN A. " 5 µg/ml polydeoxyadenylic acid. " 100 µg/ml polyadenylic acid. " 0.05 pM/ml Randomer O ligonucleotide H ybridization Probe (DuPont). " 500 µg/ml denatured and sheared salmon sperm DN A. C aution: D enature and add salm on sperm D N A to buffer shortly before hybridiz ation. " Enough deionized formamide (% dF,

as calculated by the formula below) to produce stringent hybridization conditions at 37°C [that is, to make 37°C = T m (oligonucleotide probe) – 10°C] (Long et al., 1992). C aution: % dF should not exceed 47% of the total v olum e of the hybridiz ation buffer. I f dF is less than 47% , add ddH 2O so that ddH 2O plus dF equals 47% of the v olum e. N ote: U se the follow ing form ula to calculate the form am ide concentration (% dF) in the hybridiz ation buffer for each oligonucleotide probe:

(–7.9) + 81.5 + 0.41 (% GC) – 675/L – % mismatch – (47) % dF = _______________________________________________ 0.65

*Sold under the tradename of Genius in the US.

130

where, in this case: –7.9 = 16.6 log10 [N a+] (for 2 x SSC) % GC = % [G + C] base content of the oligonucleotide L = oligonucleotide length (in bases) % mismatch = % (bases in oligonucleotide not complementary to target) 47 = hybridization temperature + 10°C (for 37°C)

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to Tissues

N ote: H ybridiz ation buffer (m inus dF, ddH 2O , and salm on sperm D N A ) can be stored at –20° C for sev eral m onths. 2 Drain 2 x SSC (Step 4, Prehybridization) !

from the slides and overlay each section with 30 µl of hybridization buffer containing 10–30 ng of digoxigeninlabeled oligonucleotide probe. ! 3 Cover sections with a 24 x 30 mm hydrophobic plastic coverslip (e.g. cut from Gel Bond Film, FMC BioProducts, Rockland, ME, USA). 4 Incubate samples at 37°C overnight in a ! humid chamber.

N egative controls ! 1 N egative sample: Tissue or cell line

known to lack the sequence of interest.

2 Technical controls: ! " Target: Digestion of mRN A with

RN ase prior to in situ hybridization (should give negative results). " H ybridization: – H ybridization with sense probe – H ybridization with irrelevant probe (e.g. probe for viral sequences) – H ybridization in the presence of excess unlabeled antisense probe (all should give negative results). " Detection: – H ybridization without probe – O mission of anti-DIG antibody (both should give negative results).

Posthybridization ! 1 Remove coverslips from sections by

immersing slides for 5–10 min in 2 x SSC.

2 In a shaking water bath at 37°C, wash !

sections as follows: " 2 x 15 min with 2 x SSC. " 2 x 15 min with 1x SSC. " 2 x 15 min with 0.25 x SSC.

Results

Immunological detection Use the same immunological detection protocol as in Procedure IIIA (for RN A probes) above. Controls for in situ hybridizations

Adequate controls must always be included to ensure the specificity of detection signals. Controls should include positive and negative samples as well as technical controls to detect false positive and negative results (H errington and McGee, 1992).

5

Positive controls ! 1 Positive sample: Tissue or cell line known

to contain mRN A of interest.

2 Technical control to test quality of tissue !

mRN A and the procedure reagents: Labeled poly(dT) probe (for oligonucleotide in situ hybridization only); labeled RN A or oligonucleotide probes complementary to abundant “housekeeping genes” (such as !-tubulin) to test quality of tissue mRN A and the procedure reagents (should give positive results).

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A

B

# Figure 1: Comparison of 35S- and digoxigenin-labeled cRNA probes for the detection of seminal vesicle secretion protein II (SVS II) mRNA in cryostat sections of the dorsolateral rat prostate. Note the equal intensity of signals in acini of the lateral lobe obtained with isotopic (Panel A) and non-isotopic (Panel B) in situ hybridization procedures. Also note the absence of signal in the coagulating glands (arrows in Panel B) which serves as an internal negative control.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to Tissues

Figure 2: SVS II mRNA detection in contiguous glands of the rat prostate with a digoxigeninlabeled antisense RNA probe. Panel A: Strong signals are present in epithelia of the ampullary gland and no signals are encountered in adjacent duct epithelia of the coagulating gland (arrow). Panel B: A higher magnification shows that the hybridization signal is restricted to the cytoplasmic portion of the glandular epithelial cells (cryostat sections).

"

A

5 B

$ Figure 3: SVS II mRNA detection in contiguous glands of the rat prostate with a digoxigeninlabeled oligonucleotide antisense probe. Hybridization signals appear to be slightly less strong than with the SVS II RNA probe (Figure 2).

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Figure 4: Detection of parathyroid hormone " (PTH) mRNA in formaldehyde-fixed, paraffinembedded tissue of a parathyroid gland with a digoxigenin-labeled antisense RNA probe. Panel A: Note the weaker signal in cells of an adenomatous lesion (upper right part) compared with the normal parathyroid tissue. Panel B: Higher magnification shows the excellent resolution of hybridization signals, which are confined to the cytoplasmic portion of cells.

B

A Figure 5: Use of a digoxigenin-labeled antisense " RNA probe and Fast Red chromogen to detect PTH mRNA in formaldehyde-fixed, paraffinembedded tissue of a parathyroid gland with chief cell hyperplasia. Panel A: PTH mRNA is marked by red precipitates. Panel B: Only weak signals are present when a sense PTH probe is used as a control.

5 A

B

A

B

Figure 6: Detection of synaptophysin mRNA in " formaldehyde-fixed, paraffin-embedded tissue of a neuroendocrine carcinoma of the gut with digoxigenin-labeled oligonucleotide probes and immunogold-silver enhancement method for visualization. Note the strong hybridization signals (consisting of silver precipitates) over tumor cells in Panel A (where an antisense probe was used) and the much weaker signal in Panel B (where the appropriate sense probe was used as a control).

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to Tissues

Reagents available from Boehringer Mannheim for this procedure

5

Reagent

Description

Cat. No.

Pack size

DIG RNA Labeling Kit* , ** , ***

For RNA labeling with digoxigenin-UTP by in vitro transcription with SP6 and T7 RNA polymerase.

1175 025

1 Kit (2x10 labeling reactions)

DIG Oligonucleotide Tailing Kit* , *** , ****

For tailing oligonucleotides with digoxigenin-dUTP.

1 417 231

1 Kit (25 tailing reactions)

DIG Nucleic Acid Detection Kit*

For detection of digoxigenin-labeled nucleic acids by an enzyme-linked immunoassay with a highly specific antiDIG-AP antibody conjugate and the color substrate NBT and BCIP.

1175 041

1 Kit

Triton® X-100

Vicous, liquid

789 704

100 ml

EDTA

Powder

808 261 808 270 808 288

250 g 500 g 1 kg

Proteinase K

Solution

1 413 783 1 373 196 1 373 200

1.25 ml 5 ml 25 ml

Pepsin

Lyophilizate

108 057 1 693 387

1g 5g

BSA

Highest quality, lyophilizate

238 031 238 040

1g 10 g

DTT

Purity: > 97%

197 777 708 984 1 583 786 708 992 709 000

2g 10 g 25 g 50 g 100 g

Tris

Powder

127 434 708 968 708 976

100 g 500 g 1 kg

RNase A

From bovine pancreas, dry powder

109 142 109 169

25 mg 100 mg

tRNA

From baker’s yeast, lyophilizate

109 495 109 509

100 mg 500 mg

Poly(A)

Polyadenylic acid

108 626

100 mg

Poly(dA)

Poly-deoxy-adenylic acid

223 581

5 A260 units

****Sold under the tradename of Genius in the US. ****EP Patent 0 324 474 granted to Boehringer Mannheim GmbH. ****Licensed by Institute Pasteur. ****EP Patents 0 124 657/0 324 474 granted to Boehringer Mannheim GmbH.

134

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to Tissues

Acknowledgments

References

I am grateful to Ph. U. H eitz and J. Roth for general support; to A. A. Long, H . J. Wolfe, S. P. N aber, and W. Membrino for technical advice; to M. Machado, X. Matias-Guiu, F. B. Merck, I. Leav, and P. Saremaslani for technical support; and to N . Wey and H . N ef for photographic reproductions. The projects – during which the protocols have been established – were supported in part by the Julius Müller-Grocholski Cancer Research Foundation, Zürich Switzerland.

Herrington, C. S.; McGee, J. O. (1992) Principles and basic methodology of DNA/RNA detection by in situ hybridization. In: Herrington, C. S.; McGee, J. O. (Eds.) Diagnostic molecular pathology: A practical approach. New York: IRL Press, Vol. 1, pp. 69–102. Komminoth, P. (1992) Digoxigenin as an alternative probe labeling for in situ hybridization. Diagn. Mol. Pathol. 1, 142–150. Komminoth, P.; Merk, F. B.; Leav, I.; Wolfe, H. J.; Roth, J. (1992) Comparison of 35S- and digoxigeninlabeled RNA and oligonucleotide probes for in situ hybridization. Expression of mRNA of the seminal vesicle secretion protein II and androgen receptor genes in the rat prostate. Histochemistry 98, 217–228. Komminoth, P.; Roth, J.; Saremaslani, P.; Schrödel, S., Heitz, P. U. (1995) Overlapping expression of immunohistochemical markers and synaptophysin mRNA in pheochromocytomas and adrenocortical carcinomas. Implications for the differential diagnosis of adrenal gland tumors. Lab. Invest. 72, 424–431. Long, A. A.; Mueller, J.; Andre-Schwartz, J.; Barrett, K.; Schwartz, R., Wolfe, H. J. (1992) High-specificity in situ hybridization: Methods and application. Diagn. Mol. Pathol. 1, 45–57. Naber, S.; Smith, L.; Wolfe, H. J. (1992) Role of the frozen tissue bank in molecular pathology. Diagn. Mol. Pathol. 1, 73–79. Sambrook, J.; Fritsch, E.; Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

5

Valentino, K. L.; Eberwine, J. H.; Barchas, J. D. (1987) In situ Hybridization: Applications To Neurobiology. Oxford, England: Oxford University Press. Young, I. D.; Ailles, L.; Deugau, K.; Kisilevsky, R. (1991) Transcription of cRNA for in situ hybridization from Polymerase Chain Reaction-amplified DNA. Lab. Invest. 64, 709–712.

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135

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to Tissues

Detection of mRNA on paraffin embedded material of the central nervous system with DIG-labeled RNA probes H . Breitschopf and G . Suchanek , R esearch U nit for Experim ental N europathology, A ustrian A cadem y of Sciences, V ienna, A ustria.

The following protocol was developed to show mRN A in paraffin embedded material of the central nervous system. It also allows double staining of protein and mRN A. This method is sensitive enough to show mRN A expressed at very low levels (Breitschopf et al., 1992).

I. Probe preparation ! 1 Prepare plasmids by standard methods

(Sambrook et al., 1989). 2 Prepare RN A probes from the plasmids ! and label them with digoxigenin (DIG) either according to the methods in Chapter 4 of this manual or according to the instructions in the DIG RN A Labeling Kit*. ! 3 Remove unincorporated nucleotides from the labeled probes by passing the labeling mixture over a Q uick Spin™ column for radiolabeled RN A preparation (Sephadex® G-50). C aution: O m ission of this colum n step m ay lead to back ground problem s.

5

4 Determine the amount of DIG-labeling !

with a dot blot according to the method in Chapter 4 of this manual.

II. Tissue preparation ! 1 U se samples fixed by standardized

*Sold under the tradename of Genius in the US.

136

methods, such as: " Perfusion-fixed samples. C aution: Before using perfusion-fixed samples, treat them further by immersion-fixing for 3–4 h at room temperature in 100 mM phosphate buffer containing 4% paraformaldehyde. N ote: Sam ples fixed w ith 4% paraform aldehyde giv e the best results in this procedure. " Routinely fixed autopsy samples. " Samples fixed with 2.5% glutaraldehyde. 2 After fixation, wash the samples in ! phosphate buffered saline (PBS). ! 3 Embed the fixed samples in paraffin.

4 Coat slides with a 2% solution of !

3-aminopropyltriethoxy-silane (Sigma) in acetone, then air dry them. C aution: Perform Step 4 under R N asefree conditions. 5 Cut 4 µm thick sections from the fixed !

samples with disposable knifes and attach them to the prepared slides. C aution: Perform Step 5 under R N asefree conditions. 6 Dry the sections overnight at 55°C. ! ! 7 Store the sample slides at room temper-

ature until they are used.

III. Pretreatment of sections C aution: Perform pretreatm ent and hybridiz ation steps under R N ase-free conditions. Prepare all solutions and buffers for these steps w ith D EPC -treated w ater (Sam brook et al., 1989). Bak e all glassw are for 4 h at 180° C . A ssum e that disposable plasticw are is R N ase-free. ! 1 Dewax slides extensively by treating with

xylene (preferably overnight) and a graded series of ethanol solutions. 2 To preserve the mRN A during the ! following procedure, fix the sections once again with 4% paraformaldehyde (in 100 mM phosphate buffer) for 20 min. ! 3 Rinse the sections 3–5 times with TBS buffer [50 mM Tris-H Cl (pH 7.5); 150 mM N aCl]. 4 Treat the sections for 10 min with ! 200 mM H Cl to denature proteins. 5 Rinse the sections 3–5 times with TBS. ! 6 To reduce nonspecific background (H a! yashi et al., 1978), incubate the sections for 10 min on a magnetic stirrer with a freshly mixed solution of 0.5% acetic anhydride in 100 mM Tris (pH 8.0). ! 7 Rinse the sections 3–5 times with TBS. 8 Treat the sections for 20 min at 37°C with ! Proteinase K (10–500 µg per ml in TBS which contains 2 mM CaCl2). The concentration of Proteinase K depends upon the degree of fixation. Generally, start with 20 µg/ml Proteinase K

CONTENTS

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to Tissues

for paraformaldehyde-fixed tissue. The concentration needed for glutaraldehydefixed or overfixed tissue is generally higher than for paraformaldehyde-fixed tissue. C aution: D eterm ine the optim al concentration of Proteinase K em pirically for each type of fixation. O ne of the m ost critical steps in the w hole procedure is finding the balance betw een prior fixation and the proper concentration of Proteinase K. Proteinase K concentrations w hich are too low or too high m ay lead to false negativ e results. (See Figure 3 under “R esults”.) O ur experience show s that autolysis causes few er problem s than ov erfixation does. Ev en after 30 hours autolysis, w e could dem onstrate m R N A . (See Figure 4 under “R esults”.) I n ov erfixed m aterial, w e w ere not alw ays successful in dem onstrating m R N A , ev en w hen w e increased Proteinase K to v ery high concentrations (up to 500 m g/ m l). ! 9 Rinse the sections 3–5 times with TBS. 10 Incubate the sections at 4°C for 5 min !

with TBS (pH 7.5) to stop the Proteinase K digestion. C aution: D o not postfix the sections w ith paraform aldehyde after protease digestion, since this reduces signal intensity. 11 Dehydrate the sections in a graded series !

of ethanol solutions (increasing concentrations of ethanol). ! 12 Rinse the sections briefly with chloroform. N ote: A t this point, you m ay, if necessary, store the sections under dust- and R N ase-free conditions for sev eral days.

IV. Hybridization C aution: Perform the hybridiz ation step under R N ase-free conditions. Prepare all solutions and buffers for this step w ith D EPC -treated w ater (Sam brook et al., 1989). Bak e all glassw are for 4 h at 180° C . A ssum e that disposable plasticw are is R N ase-free. ! 1 Place the sections in a humid chamber at

55°C for 30 min.

2 Prepare a hybridization buffer by mixing !

the following components, then vortexing vigorously: " 2 x SSC (Sambrook et al., 1989). " 10% dextran sulfate.

CONTENTS

INDEX

" 0.01% sheared salmon sperm DN A

(Sambrook et al., 1989).

" 0.02% SDS. " 50% formamide.

C aution: T he concentration of dextran sulfate is critical. Without appropriate am ounts of dextran sulfate, the m ethod loses sensitiv ity. H ow ev er, excessiv e concentrations of dextran sulfate causes higher v iscosity of the hybridiz ation buffer. To prev ent unev en distribution of the probe and unev en signals, alw ays v ortex the hybridiz ation buffer extensiv ely. ! 3 Dilute the labeled antisense RN A probe

to an appropriate degree in hybridization buffer. The diluted probe solution may contain as much as 1 part labeled probe to 4 parts hybridization buffer. N ote: T he am ount of probe needed depends upon the degree of probe labeling (as determ ined in Procedure I , Step 4 abov e). G enerally, use the low est probe concentration that giv es optim al response in that dot blot procedure. Example: I n a serial dilution of the probe on a dot blot , if a 1:160 dilution giv es a significantly stronger response than a 1:320 dilution, perform the initial hybridiz ation experim ents w ith both a 1:200 and a 1:300 dilution of the probe. 4 Pipette the diluted !

antisense probe solution onto each section at a volume of 10 µl/cm 2. Cover with a coverslip. N ote: I n our experience, a prehybridiz ation step (w ith hybridiz ation buffer alone) did not im prov e results.

5

5 As a control serve sections treated in the !

same way whereby in steps 3 and 4 a labeled sense RN A probe is used. C aution: C ontrol hybridiz ation w ith sense probes is necessary to prov e the specificity of the reaction. H ow ev er, hybridiz ation w ith sense probes som etim es giv es unexpected hybridiz ation signals or back ground staining. 6 Place the slides on a hot plate at 95°C for !

4 min. N ote: T his step increases the signal from R N A / R N A hybrids. ! 7 Incubate the slides in a humid chamber for 4–6 h at 55°–75°C. N ote: H ybridiz ation specificity can be increased by increasing the tem perature of hybridiz ation, if necessary, as high as 75° C . I ncreasing the tem perature can help to differentiate m R N A s of highly hom ologous proteins (Taylor et al., 1994). 137

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to Tissues

V. Washes and detection of mRNA N ote: Stringency w ashings, often described as a m ethod to rem ov e nonspecifically bound probes, are helpful in reducing diffuse back ground staining, but do not significantly im prov e the specificity of the hybridiz ation. ! 1 Incubate the slides in 2 x SSC overnight.

N ote: T he cov erslips w ill float off during this incubation. 2 Wash the slides as follows: ! " 3 x 20 min at 55°C with 50% deionized

formamide in 1x SSC.

" 2 x15 min at room temperature with

1x SSC . ! 3 Rinse the sections 3–5 times with TBS. 4 Incubate the slides for 15 min with ! blocking mixture (blocking reagent containing 10% fetal calf serum). 5 Incubate the slides for 60 min with ! alkaline-phosphatase-conjugated antiDIG antibody [diluted 1: 500 in blocking mixture (from Step 4 above)]. 6 Rinse the sections 3–5 times with TBS. ! ! 7 Prepare N BT/BCIP color reagent as recommended by the manufacturer. 8 In a Coplin jar in the refrigerator, ! incubate the sections with color reagent until sufficient color develops. N ote: I ncubation can be extended up to 120 h if the color reagent is replaced ev ery tim e it precipitates or changes color.

5

! 9 Stop the color reaction by rinsing the

slides several times with tap water.

10 Finally, rinse the slides with distilled !

water.

11 Mount the slides directly with any water!

soluble mounting medium. N ote: O ptionally, counterstain the slides or use im m unocytochem istry (as in Procedure V I below ) to v isualiz e proteins on the slides.

VI. Detection of proteins by immunocytochemistry N ote: T his protocol describes a triple A PA A P (alk aline phosphatase anti-alk aline phosphatase) reaction (Vass et al., 1989), w hich allow s v isualiz ation of proteins on the sam e slide w ith the m R N A . See Figure 1 under “R esults” for an exam ple of double staining obtained w ith this technique.

138

! 1 Incubate slide overnight at 4°C with

primary antibody (either polyclonal or mouse monoclonal), diluted as necessary in TBS containing 10% fetal calf serum (TBS-FCS). Example: T he polyclonal antibody for proteolipid protein used in Figure 1 w as diluted 1:1000. 2 Rinse the slide 3–5 times with TBS at !

room temperature. N ote: Perform Step 2 as w ell as all the rem aining incubation and w ash steps of Procedure V I at room tem perature. ! 3 Depending upon the type of primary

antibody used in Step 1, do one of the following: " If the primary antibody was a polyclonal antibody from rabbit, incubate the slide for 60 min with anti-rabbit serum from mouse [Dakopatts], diluted 1:100 in TBS-FCS, then rinse the slide 3–5 times with TBS. Go to Step 4. " If the primary antibody was a mouse monoclonal antibody, skip this step and go to Step 4. 4 Incubate slide for 60 min with anti-mouse !

serum (from rabbit [Dakopatts], diluted 1:100 in TBS-FCS), then rinse the slide 3–5 times with TBS. 5 Incubate slide for 60 min with APAAP ! complex (mouse) (diluted 1:100 in TBSFCS), then rinse the slide 3–5 times with TBS. 6 Incubate slide for 30 min with anti-mouse ! serum (from rabbit) (diluted 1:100 in TBS-FCS), then rinse the slide 3–5 times with TBS. ! 7 Incubate slide for 30 min with APAAP complex (mouse) (diluted 1:100 in TBSFCS), then rinse the slide 3–5 times with TBS. 8 Repeat Steps 6 and 7. ! N ote: R epeating the A PA A P steps w ill enhance the signal. ! 9 Prepare APAAP substrate by dissolving

200 mg naphthol-ASMX-phosphate, 20 ml dimethylformamide, and 1 ml of 1 M levamisole (all from Sigma) in 980 ml Tris-H Cl (pH 8.2). 10 Dissolve 50 mg Fast Red TR Salt in 50 ml ! of APAAP substrate, then filter the solution. 11 In a Coplin jar at room temperature, ! incubate the slides with the Fast RedAPAAP substrate solution until the color develops.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to Tissues

N ote: I f you substitute PA P com plex for A PA A P com plex, m odify the abov e procedure as follow s:

PFA

GA

" Perform all rinses and all dilutions w ith

PBS rather than T BS.

" For the color reaction, use filtered dia-

m inobenz idine reagent (50 m g diam inobenz idine dissolv ed in 100 m l PBS w hich contains 0.01% H 2O 2) For an exam ple of im m unostaining w ith PA P com plex, see Figure 2 under “R esults”.

0.002%

Results

0.005%

#

Figure 1: Double staining of mRNA and protein (APAAP method) in a normal rat brain. The mRNA (black-blue) for proteolipid protein (PLP) was detected by the procedure described in the text and stained with NBT/BCIP color reagent. Proteolipid protein (red) was detected with a 1:1000-diluted rabbit polyclonal antibody and visualized with APAAP and Fast Red TR Salt.

5

0.02%

0.05%

#

Figure 2: Double staining of mRNA and protein (PAP method) in a normal rat brain. The experiment is identical to that in Figure 1, except PLP (brown) was visualized with PAP and diaminobenzidine reagent.

CONTENTS

INDEX

#

a

b

Figure 3: Effect of paraformaldehyde and glutaraldehyde fixation on in situ hybridization of mRNA. PLP mRNA was detected in sections of rat spinal cord which had been fixed by different methods. Panel a: Sections were fixed with 4% paraformaldehyde and then treated with different concentrations of Proteinase K. From top to bottom the Proteinase Kconcentrations were: 0.002%, 0.005%, 0.02%, 0.05%. Panel b: Sections were fixed in 2.5% glutaraldehyde, then treated with the same concentrations of Proteinase K as in Panel a.

139

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to Tissues

Figure 4: Effect of autolysis and " overfixation on in situ hybridization of mRNA and immunocytochemical staining. In spite of pronounced autolytic changes that occur postmortem, a good PLP mRNA signal can be seen in human brain autopsy samples (Panel a). Although in well preserved and fixed experimental tissue (Panel b) both, the signal for in situ hybridization as a well as the signal for immunocytochemistry is much more distinct. Protein (red) and mRNA (blue-black) were stained as in Figure 1.

References Breitschopf, H; Suchanek, G; Gould, R. M.; Colman, D. R.; Lassmann, H. (1992) In situ hybridization with digoxigenin-labeled probes: Sensitive and reliable detection method applied to myelinating rat brain. Acta Neuropathol. 84, 581–587. Hayashi, S; Gillam, I. C.; Delaney, A. D.; Tener, G. M. (1978) Acetylation of chromosome squashes of Drosophila melanogaster decreases the background in autoradiographs from hybridization with 125I-labeled RNA. J. Histochem. Cytochem. 26, 677–679. Sambrook, J; Fritsch, E. F.; Maniatis, T. (1989) Molecular cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press.

Acknowledgments

Taylor, V.; Miescher, G. C.; Pfarr, S.; Honegger, P.; Breitschopf, H.; Lassmann, H.; Steck, A. J. (1994) Expression and developmental regulation of Ehk-1, a neuronal Elk-like receptor tyrosine kinase in brain. Neuroscience 63, 163–178.

The rabbit polyclonal antibody against proteolipid protein (PLP) was a gift from Dr. S. Piddlesden, University of Cardiff, UK.

Vass, K.; Berger, M. L.; Nowak, T. S. Jr.; Welch, W. J.; Lassmann, H. (1989) Induction of stress protein HSP 70 in nerve cells after status epilepticus in the rat. Neurosci. Lett. 100, 259–264.

b

Reagents available from Boehringer Mannheim for this procedure

5

Reagent

Description

Cat. No.

Pack size

DIG RNA Labeling Kit* , ** , ***

For RNA labeling with digoxigenin-UTP by in vitro transcription with SP6 and T7 RNA polymerase.

1175 025

1 Kit (2x10 labeling reactions)

Proteinase K

Lyophilizate

161 519 745 723 1 000 144 1 092 766

25 mg 100 mg 500 mg 1g

Tris

Powder

127 434 708 968 708 976

100 g 500 g 1 kg

Blocking Reagent

Powder

1 096 176

50 g

Anti-DIG-AP

Fab Fragments from sheep

1 093 274

150 U (200 µl)

NBT/BCIP

Stock solution

1 681 451

8 ml

***Sold under the tradename of Genius in the US. ***EP Patent 0 324 474 granted to Boehringer Mannheim GmbH. ***Licensed by Institute Pasteur.

140

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to Tissues

RNA-RNA in situ hybridization using DIG-labeled probes: the effect of high molecular weight polyvinyl alcohol on the alkaline phosphatase indoxyl-nitroblue tetrazolium reaction M arc D eBlock and D irk D ebrouw er, Plant G enetic System s N .V., G ent, Belgium .

N ote: T he follow ing article is reprinted w ith the perm ission of A cadem ic Press (C opyright ® 1993 by A cadem ic Press, I nc.) and H arcourt Brace & C om pany and is based on the original article: D eBlock , M ., D ebrouw er, D . (1993) R N A -R N A in situ hybridiz ation using digoxigenin-labeled probes: the use of high m olecular w eight polyv inyl alcohol in the alk aline phosphatase indoxyl-nitroblue tetrazolium. Analytical Biochemistry 216; 88–89. The indoxyl-nitroblue tetrazolium (BCIPN BT) reaction is relatively slow. During the reaction, intermediates (indoxyls) diffuse away into the medium, making it difficult to localize the site of hybridization (Van N oorden and Jonges, 1987) and reducing the hybridization signal. In this paper, an improved nonradioactive RN A-RN A in situ hybridization protocol using alkaline phosphatase-conjugated digoxigenin- (DIG-) labeled probes is presented. The addition of polyvinyl alcohol (PVA) of high molecular weight (40–100 kD) to the BCIP-N BT detection system enhances the alkaline phosphatase reaction and prevents diffusion of reaction intermediates, resulting in a twentyfold increase in sensitivity without increasing the background. Due to the more localized precipitation of the formazan, the site of hybridization can be determined more precisely.

CONTENTS

INDEX

I. Fixation, dehydration, and embedding Follow the protocol described by Jackson (1992) to fix, dehydrate, and embed the tissue in paraffin, with the following modifications: ! 1 For fixation, prepare either of the follow-

ing solutions: " 100 mM phosphate buffer, pH 7, containing 0.25% gluteraldehyde and 4% freshly depolymerized paraformaldehyde. " Formalin-acetic acid (50% ethanol; 10% formalin, containing 37% formaldehyde; 5% acetic acid). 2 Using either of the solutions from ! Step 1, fix the tissue for 4 h at room temperature. During the fixation: " Vacuum infiltrate the tissue (with a water aspirator) for 10 min once an hour. " After each vacuum infiltration, renew the fixative solution. ! 3 Depending on the fixative used in Step 2, wash the fixed tissue as follows: " If gluteraldehyde-paraformaldehyde was the fixative, wash the fixed tissue 2 x 30 min with 100 mM phosphate buffer, pH 7. " If formalin-acetic acid was the fixative, wash the fixed tissue 2 x 30 min with 50% ethanol. 4 Dehydrate the tissue by incubating in the ! following series of ethanol solutions: " Either 90 min (after gluteraldehydeparaformaldehyde fixation) or 30 min (after formalin-acetic acid fixation) at room temperature in 50% ethanol. " 90 min at room temperature in 70% ethanol. " O vernight at 4°C in 85% ethanol. " 3 x 90 min at room temperature in 100% ethanol.

5

141

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to Tissues

Polymer

Molecular weight

Concentration b

Effect on the alkaline phosphatase BCIP-N BT reaction e

PVP

15 kD 25 kD 44 kD

10, 20, 30% 10, 20, 30% 10, 20, 30%

Autoreduction of N BT

PEG

6 kD 20 kD

10, 20, 30% 10, 20, 30%

N o enhancement (0.01 mM formazan)

PVA

10 kD

10, 20% c

Approx. 4-fold enhancement (0.04 mM formazan) Approximately 6- to 8-fold enhancement (0.06 to 0.08 mM formazan)

40 kD 70–100 kD

10% d 10% d

# Table 1: Influence of polymers on the alkaline phosphatase BCIP-NBT reactiona. a Each reaction was done with a total volume of 2 ml BCIP-NBT reaction mixture in a test tube. The BCIP-NBT reaction mixture was as described in the text, except that it contained 7.5 X 10 –3 units alkaline phosphatase/ml and the polymer indicated in the table. Incubation was for 30 min at 24° C. b The polymers were dissolved in the alkaline phosphatase reaction buffer in concentrations between 10% and 30%. The concentrations are given as a percentage of weight to volume. c A solution of 30% 10 kD PVA was too viscous. d Solutions of 20 or 30% 40 kD PVA and 20 or 30% 70–100 kD PVA were too viscous. e The amount of formazan formed was measured at 605 nm (Eadie et al., 1970). The enhancement factor is expressed with respect to the controls (no polymer added). If no polymer was added, about 0.01 mM formazan was formed. a

b

c

5

#

Figure 1: The influence of PVA on the alkaline phosphatase BCIP-NBT reaction in in situ hybridizations. The hybridizations were carried out on 10 µm paraffin sections of stigmas and styles of tobacco with antisense and sense DIG-labeled RNA probes of pMG07 (de S. Goldman et al., 1992). C, cortex tissue; TT, transmitting tissue; V, vascular tissue. Bars = 200 µm. Panel a: Hybridization with antisense probe. Twenty percent 10 kDPVA was added to the alkaline phosphatase reaction buffer. Development was done for 4 h. Panel b: Hybridization with antisense probe. Ten percent 70–100 kD PVA was added to the alkaline phosphatase reaction buffer. Development was done for 2 h. Panel c: Hybridization with sense RNA probe. Ten percent 70–100 kD PVA was added to the alkaline phosphatase reaction buffer. Development was done for 20 h.

144

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to Tissues

References DeBlock, M.; Debrouwer, D. (1993) RNA-RNA in situ hybridization using digoxigenin-labeled probes: the use of high molecular weight polyvinyl alcohol in the alkaline phosphatase indoxyl-nitroblue tetrazolium reaction. Anal. Biochem. 215, 86–89. de S. Goldman, M. H.; Pezzotti, M.; Seurinck, J.; Mariani, C. (1992) Developmental expression of tobacco pistil-specific genes encoding novel extensin-like proteins. Plant Cell 4, 1041–1051. Eadie, M. J.; Tyrer, J. H.; Kukums, J. R.; Hooper, W. D. (1970) Histochemie 21, 170–180. Jackson, D. (1991) In situ hybridization in plants. In: Gurr, S. J.; McPherson; Bowles, D. J. (Eds.) Molecular Plant Pathology, A Practical Approach. Oxford, England: Oxford University Press, Vol. 1, pp. 163–174. Van Noorden, C. J. F.; Jonges, G. N. (1987) Quantification of the histochemical reaction for alkaline phosphatase activity using the indoxyltetranitro BT method. Histochem. J. 19, 94–102.

Reagents available from Boehringer Mannheim for this procedure Reagent

Description

Cat. No.

Pack size

DIG RNA Labeling Kit* , ** , ***

For RNA labeling with digoxigenin-UTP by in vitro transcription with SP6 and T7 RNA polymerase.

1175 025

1 Kit (2x10 labeling reactions)

Proteinase K

Lyophilizate

161 519 745 723 1 000 144 1 092 766

25 mg 100 mg 500 mg 1g

tRNA

From baker’s yeast, lyophilizate

109 495 109 509

100 mg 500 mg

DNA from herring sperm

Lyophilized, sodium salt

223 646

1g

DTT

Purity: > 97%

197 777 708 984 1 583 786 708 992 709 000

2g 10 g 25 g 50 g 100 g

RNase A

Powder

109 142 109 169

25 mg 100 mg

RNase Inhibitor

From human placenta

799 017 799 025

2,000 units 10,000 units

DIG Nucleic Acid Detection Kit*

For detection of digoxigenin-labeled nucleic acids by an enzyme-linked immunoassay with a highly specific antiDIG-AP antibody conjugate and the color substrates NBT and BCIP.

1175 041

1 Kit

5

***Sold under the tradename of Genius in the US. ***EP Patent 0 324 474 granted to Boehringer Mannheim GmbH. ***Licensed by Institute Pasteur.

CONTENTS

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to Tissues

II. Sectioning ! 1 Cut the paraffin-embedded tissues in

10 µm sections. 2 Attach the sections at 75°C for 1 h to ! slides that have been treated with Vectabond (Vector Laboratories, Burlingame, CA, U.S.A.).

III. Prehybridization treatments ! 1 Dewax and hydrate the sections as

previously described (Jackson, 1992).

2 Incubate the sections with a Proteinase K !

solution (100 mM Tris, pH 7.5; 50 mM EDTA; 2 µg/ml Proteinase K) for 30 min at 37°C. ! 3 After the Proteinase K treatment, wash the slides 2 x in phosphate buffered saline (PBS). 4 Dehydrate the sections in ascending ! concentrations of ethanol as previously described (Jackson, 1991).

IV. Hybridization ! 1 Label the probe RN A with DIG-UTP

according to the procedures given in Chapter 4 of this manual. 2 Reduce the length of the probe to ! approximately 200 bases as follows: " To 50 µl of labeled probe RN A in a microcentrifuge tube, add 30 µl 200 mM N a2CO 3 and 20 µl 200 mM N aH CO 3. " H ydrolyze the probe at 60°C for t min, where: t = (L 0 – L f) / (K · L 0 · L f)

5

L 0 = starting length of probe RN A (in kb) L f = length of probe RN A (in kb) (In this case, L f = 0.2 kb.) K = rate constant (In this case, K = 0.11 kb/min.) t = hydrolysis time in min ! 3 After hydrolysis, purify the probe RN A

as follows: " Add the following to the hydrolyzed probe solution: – 5 µl 10% acetic acid – 11 µl 3 M sodium acetate (pH 6.0) – 1 µl of a 10 mg/ml tRN A stock – 1.2 µl 1 M MgCl2 – 300 µl (about 2.5 volumes) cold ethanol

142

" Incubate 4–16 h at –20°C. " Centrifuge in a microcentrifuge for 15

min at 4°C to pellet the RN A.

" Discard the supernatant and dry the

RN A pellet in a vacuum desiccator.

" Resuspend labeled probe RN A in

DEPC-treated water at 10–50 µg/ml.

4 Prepare a hybridization mixture con!

taining the following: " 50% deionized formamide. " 2.25 x SSPE (300 mM N aCl; 20 mM N aH 2PO 4; 2 mM EDTA; pH 7.4). " 10% dextran sulfate. " 2.5 x Denhardt’s solution. " 100 µg/ml sheared and denatured herring sperm DN A. " 100 µg/ml tRN A. " 5 mM DTT. " 40 units/ml RN ase inhibitor. " 0.2–1.0 µg/ml hydrolyzed and denatured probe (200 bases long). 5 Cover each section with 250–500 µl of !

hybridization mixture (depending on the size of the section) and incubate in a humidified box at 42°C overnight. N ote: D o not use a cov erslip during the hybridiz ation incubation. 6 After hybridization, wash the slides as !

follows: " 5 min at room temperature with 3 x SSC. N ote: 1 x SSC contains 150 m M N aC l, 15 m M N a-citrate; pH 7. " 5 min at room temperature with N TE

(500 mM N aCl, 10 mM Tris-H Cl, 1 mM EDTA; pH 7.5). ! 7 To remove unhybridized single-stranded RN A probe, put slides into a humidified box and cover each section with 500 µl of N TE buffer containing 50 µg/ ml RN ase A. Incubate for 30 min at 37°C. 8 After RN ase treatment, wash the slides ! 3 x 5 min at room temperature with N TE. ! 9 To remove nonspecifically hybridized probe, wash the slides as follows: " 30 min at room temperature with 2 x SSC. " 1 h at 57°C with 0.1 x SSC.

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V. Detection of DIG-labeled hybrids ! 1 Incubate the slides first with blocking

solution, then with blocking solution containing 1.25 units/ml of alkaline phosphatase-conjugated anti-DIG Fab fragments as recommended in the pack insert of the DIG N ucleic Acid Detection Kit*. 2 After the antibody incubation, wash the ! slides to remove unbound antibody as recommended in the Boehringer Mannheim DIG/Genius™ System User’s Guide for Membrane H ybridization. ! 3 Prepare the BCIP-N BT-PVA color development solution as follows: " Prepare a Tris-N aCl-PVA stock solution by dissolving 10% (w/v) polyvinyl alcohol [PVA, either 40 kD or 70–100 kD (Sigma)] at 90°C in 100 mM Tris-H Cl (pH 9) containing 100 mM N aCl. " Cool the Tris-N aCl-PVA stock solution to room temperature. " Add MgCl2, BCIP, and N BT to the Tris-N aCl-PVA stock to produce a final color development solution that contains: – 5 mM MgCl – 0.2 mM 5-bromo-4-chloro-3-indolyl phosphate (BCIP) – 0.2 mM nitroblue tetrazolium salt (N BT). 4 After the washes in Step 2, perform the ! visualization step as follows: " Place the slides in 30 ml of BCIPN BT-PVA color development solution in a vertical staining dish suited for eight slides. " Incubate the slides in the color development solution for 2–16 h at 30°C. " Monitor color formation visually. 5 When the color on each slide is optimal, ! stop the color reaction by washing the slide 3 x 5 min in distilled water.

6 Dehydrate the sections by incubating the !

slides without shaking in the following ethanol solutions: " 15 s in 70% ethanol. " 2 x15 s in 100% ethanol. ! 7 Air dry the slides and mount them with Eukitt (O . Kindler GmbH , FRG). 8 Examine the sections with a Dialux ! 22 microscope (Leitz, Wetzlar, FRG) equipped with N ormaski differential interference contrast.

Results and discussion The direct influence of the polymers on the BCIP-N BT alkaline phosphatase reaction in a test tube is shown in Table 1. From these results it is clear that polyvinyl alcohol was the only polymer that enhanced formazan formation in the alkaline phosphatase reaction. This enhancement was even more pronounced in in situ hybridization experiments. Figure 1 shows the results of an in situ hybridization in which sections of tobacco pistils were hybridized with an antisense RN A probe from a pistil-specific cDN A clone (de S. Goldman et al., 1992). After three hours development no hybridization could be detected if no PVA had been added to the reaction buffer. A clear but still weak signal was visible after three h if 20% PVA (molecular weight, 10 kD) was used (Figure 1a). H owever, with 10% PVA (molecular weight, 70–100 kD), a strong hybridization signal appeared in the transmitting tissue of the pistil after a few h (Figure 1b). In the control with the sense RN A probe no signal could be detected, even after 24 h of development (Figure 1c).

5

*Sold under the tradename of Genius in the US.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to Tissues

Detection of neuropeptide mRNAs in tissue sections using oligonucleotides tailed with fluorescein-12-dUTP or DIG-dUTP D epartm ent of C ytochem istry and C ytom etry, U niv ersity of L eiden, T he N etherlands. The protocol given below has been developed with the neuropeptidergic system of the pond snail Lymnaea stagnalis. More information concerning the application and in situ hybridization methodology can be found in Van Minnen et al. (1989), Dirks et al. (1988), Dirks et al. (1990), and Dirks et al. (1991).

I. Tissue preparation ! 1 Dissect the tissue, embed in O .C.T.

compound (Miles Scientific, USA), and freeze in liquid nitrogen. 2 Prepare sections as follows: ! " Cut cryostat sections of 8 µm. " Mount on poly-L-lysine-coated slides. " Air dry. ! 3 After mounting, do the following: " Fix the sections for 30 min at 4°C with modified Carnoy’s [2% formalin, (from 37% stock), 75% ethanol, 23% acetic acid]. " Rinse the slides with water. " Dehydrate the sections.

5

! 1 Perform the hybridization as follows: " Prepare hybridization mixture [25%

formamide; 3 x SSC; 0.1% polyvinylpyrrolidone; 0.1% ficoll, 1% bovine serum albumin; 500 µg/ml sheared salmon sperm DN A; 500 µg/ml yeast RN A]. Note: 1x SSC contains 150 mM sodium chloride, 15 mM sodium citrate; pH 7.0. " To the cryostat sections, add hybridi-

zation mixture containing 1 ng/µl labeled probe. " Incubate for 2 h at room temperature. N ote: I f the probe contains a fluorochrom e label, perform the hybridiz ation incubation in the dark . 2 After hybridization, do the following: ! " Rinse the section 3 times in 4 x SSC at

room temperature.

" Dehydrate the sections.

N ote: I f the probe contains a fluorochrom e label, perform the w ashes in the dark .

IV. Hybrid detection II. Probe preparation Synthesize and purify the oligonucleotides according to routine procedures. Tail the oligonucleotide with DIG-dUTP or fluorescein-dUTP according to the procedures given in Chapter 4, but without using dATP. N ote: I f you use fluorescein-dU T P, add to the labeling m ixture equal am ounts of unm odified dT T P and fluorescein-dU T P, then carry out the labeling procedure in the dark .

III. In situ hybridization N ote: 18-m ers are used in this study. For oligonucleotides w ith other lengths and/ or com position, you m ay hav e to alter the stringency of hybridiz ation by changing the form am ide concentration or the hybridiz ation tem perature listed below.

146

Depending on the label used on the probe, follow one of these procedures: " If sections are hybridized with fluoresceinlabeled oligonucleotides: " Mount sections in PBS/glycerol (1:9; v/v) containing 2.3% 1,4-diazabicyclo(2,2,2)-octane (DABCO , from Sigma) and 0.1 µg/µl 4',6'-diamidino-2phenylindole (DAPI). " Evaluate under a fluorescence microscope. " If sections are hybridized with DIGlabeled oligonucleotides: " Make a 1:250 dilution of fluoresceinconjugated anti-DIG antibody (from sheep) in incubation buffer [100 mM Tris-H Cl, pH 7.4; 150 mM N aCl; 1% blocking reagent]. " O verlay the sections with the diluted antibody in incubation buffer. " Incubate sections at room temperature for 30 min.

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" Rinse sections 3 x 5 min in Tris-N aCl

[100 mM Tris, pH 7.4; 150 mM N aCl].

" Mount and evaluate as for fluorescein-

labeled oligonucleotides.

Results # Figure 2: Indirect detection of

caudodorsal cells with DIG-labeled CDCH-I and sheep anti-DIG-fluorescein conjugate. The same cells are positive, but the hybridization signal is clearly more intense than that obtained with the direct technique (Figure 1).

$

Figure 1: Direct detection of caudodorsal cells with fluorescein-labeled CDCH-I.

References Dirks, R. W.; van Gijlswijk, R. P. M.; Vooijs, M. A.; Smit, A. B.; Bogerd, J.; Van Minnen, J.; Raap, A. K.; Van der Ploeg, M. (1991) 3'-end fluorochromized and haptenized oligonucleotides as in situ hybridization probes for multiple, simultaneous RNA detection. Exp. Cell Res. 194, 310–315. Dirks, R. W.; van Gijlswijk, R. P. M.; Tullis, R. H.; Smit, A. B.; Van Minnen, J.; Van der Ploeg, M.; Raap, A. K. (1990) Simultaneous detection of different mRNA sequences coding for neuropeptide hormones by double in situ hybridization using FITCand biotin-labeled oligonucleotides. J. Histochem. Cytochem. 38, 467–473.

Dirks, R. W.; Raap, A. K.; Van Minnen, J.; Vreugdenhil, E.; Smit, A. B.; Van der Ploeg, M. (1989) Detection of mRNA molecules coding for neuropeptide hormones of the pond snail Lymnaeae stagnalis by radioactive and nonradioactive in situ hybridization: a model study for mRNA detection. J. Histochem. Cytochem. 37, 7–14. Van Minnen, J.; Van de Haar, Ch.; Raap, A. K.; Vreugdenhil, E. (1988) Localization of ovulation hormone-like neuropeptide in the central nervous system of the snail Lymnaeae stagnalis by means of immunocytochemistry and in situ hybridization. Cell Tissue Res. 251, 477–484.

5

Reagents available from Boehringer Mannheim for this procedure Reagent

Description

Cat. No.

Pack size

DIG Oligonucleotide Tailing Kit* , ** , ***

For tailing oligonucleotides with digoxigenin-dUTP.

1 417 231

1 Kit (25 tailing reactions)

Fluorescein-12-dUTP

Tetralithium salt, solution

1 373 242

25 nmol (25 µl)

tRNA

From baker’s yeast, lyophilizate

109 495 109 509

100 mg 500 mg

Anti-DigoxigeninFluorescein

Fab Fragments from sheep

1 207 741

200 µg

Anti-DigoxigeninRhodamine

Fab Fragments from sheep

1 207 750

200 µg

DAPI

Fluorescence dye for staining of chromosomes

236 276

10 mg

Blocking Reagent

Blocking reagent for nucleic acid hybridization

1 096 176

50 g

BSA

Highest quality, lyophilizate

238 031 238 040

1g 10 g

CONTENTS

INDEX

***Sold under the tradename of Genius in the US. ***EP Patents 0 124 657/0 324 474 granted to Boehringer Mannheim GmbH. ***Licensed by Institute Pasteur.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to Tissues

In situ hybridization of DIG-labeled rRNA probes to mouse liver ultrathin sections D r. D . Fischer, D . Weisenberger, and Prof. D r. U . Scheer, I nstitute for Z oology I , U niv ersity of W ürz burg, G erm any.

Thin section electron microscopy of nucleoli from mammalian cells reveals three nucleolar substructures. These are the fibrillar centers (FC), the dense fibrillar component, which forms a closely apposed layer upon the FCs, and the granular component, which constitutes the bulk of the nucleolar mass. Although there is no doubt that this ultrastructural organization somehow reflects the vectorial process of ribosome formation, the exact structurefunction relationships have been a matter of much discussion and are still largely unclear. (For a review, see Scheer and Benavente, 1990.) H ere we describe an approach to localize specific intermediates for the pre-rRN A maturation process using in situ hybridization at the ultrastructural level. This approach will allow the correlation of each pre-rRN A processing step with the defined nucleolar substructures. The procedure described can also be applied to the localization of other RN A species involved in ribosome biogenesis, such as 5S rRN A or U3 snRN A (Fischer et al., 1991).

5

4 Transfer the liver cubes to other petri !

dishes on ice, containing the fixatives (i) 4% PFA and (ii) 0.5% GA plus 4% PFA. 5 Incubate the cubes in the fixatives for ! about 2 h. 6 To remove the fixative, wash the cubes ! 3 x 5 min with ice cold PBS. IB. Dehydration of material

Transfer the object cubes to preparative glasses and dehydrate them in a series of ethanol solutions according to the following scheme: " 2 x15 min in 30% ethanol at 4°C. " 1x 30 min in 50% ethanol at 4°C. " 1x 30 min in 50% ethanol at –20°C. " 2 x 30 min in 70% ethanol at –20°C. " 2 x 30 min in 90% ethanol at –20°C. " 2 x 30 min in 96% ethanol at –20°C. " 2 x 30 min in 100% ethanol at –20°C. C aution: L eav e the objects in a sm all am ount of liquid during changes from one ethanol solution to another. T his prev ents them from drying out, especially at higher ethanol concentrations. IC. Infiltration

I. Embedding in Lowicryl K4M (according to C arlem alm and V illiger, 1989, w ith alterations) IA. Fixation of material ! 1 Prepare the following fixation solutions

and keep them on ice: " 4% paraformaldehyde (PFA) in PBS (137 mM N aCl, 2.7 mM KCl, 7 mM N a2H PO 4, 1.5 mM KH 2PO 4). " 0.5% glutaraldehyde (GA) plus 4% PFA in PBS. 2 Take a piece of freshly prepared mouse ! liver and submerge it in a petri dish filled with 4% PFA in PBS on ice. ! 3 Cut the liver into small cubes with a maximal side length of 1 mm. N ote: T he sm aller the pieces, the better reagent penetrates them in the procedure below.

148

After dehydration, ethanol must be infiltrated with the embedding medium Lowicryl K4M (Chemische Werke Lowi, Waldkraiburg, FRG). This resin tolerates a little residual water in the object and produces good results in immunocytochemistry. C aution: W hen w ork ing w ith L ow icryl K4M , perform all steps: – At low temperatures – Under the fume hood – While wearing gloves – While excluding air (oxygen) from K4M as much as possible. To exclude air from the embedding medium, do one of the following: " Fill all vessels which contain K4M with nitrogen. " Perform all steps in a closed chamber cooled to constant temperature by liquid nitrogen, thus providing a nitrogen-saturated atmosphere in the closed preparation chamber (e.g., “CS-auto”, Reichert & Jung, Cambridge Instr.).

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! 1 Prepare Lowicryl K4M as follows (to

produce a medium hard plastic): " Mix 2 g Crosslinker A, 13 g Monomer B, and 0.075 g Initiator C. " Dissolve Initiator C by gentle stirring or by bubbling dry nitrogen through the mixture. " Cool the mixture to –20°C in the freezer. 2 Perform infiltration at about –20°C with ! suitably precooled material according to the following scheme: " 1h with 100% ethanol:K4M (1:1, v/v). " 2 x1 h with K4M. " O vernight with K4M. " 2 x 3 h with K4M. ID. Embedding

Gelatin capsules (e.g. Capsulae O perculatae N o. 2) are very suitable as molds for embedding because they are transparent to UV-light and can afterwards easily be cut away. ! 1 Cool the gelatin capsules to the appropriate temperature. 2 Add a few drops of Lowicryl K4M to ! each capsule. ! 3 Drop one object cube into each capsule. Caution: Do this transfer very carefully. Do not mechanically damage the cube, allow it to dry out, or allow it to warm up. 4 Fill the capsules with the embedding !

medium.

5 Let the capsules stand for about half an !

hour to allow the air bubbles to leave. IE. Polymerization ! 1 Before switching on the UV light, turn

the temperature a bit lower to compensate for the heat from the light. 2 Induce polymerization of Lowicryl K4M ! by 360 nm long-wave UV radiation (“CS-UV”, Reichert & Jung, Cambridge Instr.) over a five day period. During the polymerization, do the following: " For about three days, keep the polymerization temperature constant at any temperature between 0°C and –40°C. " After three days, allow the temperature to rise to room temperature with about 8 K/h. N ote: For troubleshooting, see C arlem alm and V illiger (1989).

II. Sectioning The embedded mouse liver cubes can easily be recognized in the transparent resin Lowicryl K4M. Carlemalm and Villiger (1989) recommend trimming with the ultramicrotome and a glass knife to get smooth surfaces. ! 1 O btain semithin sections with a glass

knife and ultrathin sections with a diamond knife, according to standard protocols. C aution: Because of the hydrophilicity of L ow icryl K4M , be careful not to w et the surface of the block . 2 Transfer sections to 200 mesh nickel grids !

which have been coated with 0.5% parlodion. ! 3 Air dry the sections. N ote: N ow the sections are ready for hybridiz ation.

III. Probe preparation ! 1 Clone restriction fragments from mouse

rDN A.

2 Prepare in vitro transcripts from the !

rDN A clones and label them with DIGUTP according to the protocols in Chapter 4 of this manual. ! 3 Since probe length might influence the efficiency of in situ hybridization experiments, monitor transcript length by electrophoresis (in formaldehyde-containing gels), blotting onto nitrocellulose, and detection with the anti-D IG -alkaline phosphatase conjugate. 4 If necessary, reduce probe length to ! approximately 150 nucleotides by limited alkaline hydrolysis of the transcripts according to Cox et al. (1984). Briefly, the procedure is: " Incubate the transcripts with a solution of 40 mM N aH CO 3 and 60 mM N a2CO 3 at 60°C. " Calculate the Incubation time as follows:

5

L0 – Lf t = ________ k · L0 · Lf L 0 = initial length of transcript (in kb) L f = desired probe length (in kb) k = constant = 0.11 kb/min " Stop the hydrolysis by adding 3 M

sodium acetate and acetic acid.

" Precipitate the hydrolyzate with ethanol. " Dissolve the precipitate in water.

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IV. Hybridization N ote: Perform all the follow ing steps (including the hybridiz ation) in a m oist cham ber. ! 1 Prepare hybridization mixture [5 x SSC;

0.1 mg/ml tRN A; DIG-labeled antisense transcript at a final concentration of 10 ng/µl]. N ote: 1 x SSC contains 0.15 M N aC l, 0.015 M sodium citrate. 2 To monitor the specificity of binding, ! prepare a “negative” probe by substituting the sense transcript for the antisense transcript in the hybridization mixture above. ! 3 Place a droplet of the appropriate hybridization mixture onto Parafilm ®. 4 Incubate the ultrathin sections by putting ! the grid (section-side down) on top of the droplet. 5 Perform hybridization for at least 3 h at ! 65°C. 6 Wash the sections as follows, all at room ! temperature: " 3 x 5 min with 2 x SSC. " 2 x10 min with PBST (PBS containing 0.1% Tween ® 20).

V. Detection ! 1 To block nonspecific sites, incubate each

5

section for 15 min with PBST plus BG (PBST containing 1% bovine serum albumin, 0.1% CWFS-gelatin). 2 Dilute 1 nm gold-conjugated anti-DIG ! antibody 1:30 in PBST plus BG.

#

Figure 1: In situ hybridization of a digoxigeninlabeled RNA probe complementary tomost of the 28 S rRNAregion toan ultrathin section of mouse liver fixed with 3% formaldehyde and embedded in Lowicryl K4M. The nucleolus as well as the cytoplasm are clearly marked. Label is essentially absent from the mitochondria, demonstrating the specificity of the method. Cytoplasmic labeling is due to labeling of the ribosomes, especially the endoplasmatic reticulum-associated ones (Magnification: 25,200x).

150

! 3 Apply diluted antibody to section and

incubate for 1 h.

4 Wash the sections as follows: ! " 3 x 5 min with PBST. " 6 x 5 min with redistilled water. 5 Perform silver enhancement by incubating !

sectionswith developer and enhancer (from the set of silver enhancement reagents) at a ratio of 1:1 for 4–20 min (depending on the desired silver grain size). 6 Wash the sections 6 x 5 min with redis! tilled water. ! 7 Stain with 2% aqueous uranyl acetate (4 min) and Reynolds’ lead citrate for 1 min (Reynolds, 1963) according to standard procedures. 8 Evaluate the specimen in the electron ! microscope.

Results In Figure 1, the hybridized probe was detected with monoclonal anti-DIG antibody coupled to 1 nm gold particles. The signal was enhanced with silver staining for 6 min at room temperature. Silver intensification facilitates visualization of the bound hybridization probe at low magnification. The silver grains scattered throughout the nucleoplasm might indicate transport of preribosomal particles from the nucleolus to the cytoplasm. In Figure 2, a digoxigenin-labeled riboprobe complementary to the first 3 kb of the 5' region of the ETS1 was hybridized to a Lowicryl section of mouse liver and detected as described in Figure 1.

#

Figure 2: Localization of pre-rRNA molecules containing the 5' region of the ETS1. For details of the probe, see the text. The survey view shows selective labeling of the fibrillar components of the nucleolus (Magnification: 27,000x).

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References Carlemalm, E.; Villiger, W. (1989) Low temperature embedding. In: Bullock, G. R.; Petrusz, P. (Eds) Techniques Immunocytochem. 4, 29–44. Cox, K. H.; DeLeon, D. V.; Angerer, L. M.; Angerer, R. C. (1984) Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev. Biol. 101, 485–502. Fischer, D.; Weisenberger, D.; Scheer, U. (1991) Assigning functions to nucleolar structures. Chromosoma 101, 133–140. Reynolds, E. S. (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208–212. Scheer, U.; Benavente, R. (1990) Functional and dynamic aspects of the mammalian nucleolus. BioEssays 12, 14–21.

Reagents available from Boehringer Mannheim for this procedure Reagent

Description

DIG RNA Labeling Kit* , ** , ***

For RNA labeling with digoxigenin-UTP by in vitro transcription with SP6 and T7 RNA polymerase.

Tween® 20

Cat. No.

Pack size

1175 025

1 Kit (2x10 labeling reactions)

1 332 465

5x10 ml

238 031 238 040

1g 10 g

BSA

Highest quality, lyophilizate

Anti-DigoxigeninGold

Affinity purified sheep IgG to digoxigenin conjugated with ultra small gold particles (average diameter ≤ 0.8 nm).

1 450 590

1 ml

Silver Enhancement Reagents

Detection of colloidal gold particles (anti-DIG-gold) by silver deposition on the particle surface

1 465 350

1 set

5

***Sold under the tradename of Genius in the US. ***EP Patent 0 324 474 granted to Boehringer Mannheim GmbH. ***Licensed by Institute Pasteur.

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RNA in situ hybridization using DIG-labeled cRNA probes H . B. P. M . D ijk m an, S. M entz el, A . S. de Jong, and K. J. M . A ssm ann D epartm ent of Pathology, N ijm egen U niv ersity H ospital, N ijm egen, T he N etherlands. In recent years, the in situ hybridization (ISH ) technique has found widespread application in both basic science and diagnostic clinical research. The ISH technique has frequently been used to localize specific genes on metaphase chromosomes and to detect viral and bacterial genomes in infected tissues. The RN A in situ hybridization (RISH ) technique for the examination of mRN A expression has gained less attention due to the many technical problems associated with this technique. In this report, we present a step-by-step protocol for a nonradioactive RISH technique on frozen sections using digoxigeninlabeled copy RN A (cRN A) probes. We demonstrate this technique on frozen sections of mouse kidney using DIG-labeled cRN A probes for the ectoenzyme aminopeptidase A, a low-copy RN A (Assmann et al., 1992). For a high-copy RN A, we studied human psoriatic epidermis using a DIGlabeled cRN A probe for elafin/SKALP, an inhibitor of leukocyte elastase and proteinase 3 (Alkemade et al., 1994). This protocol has been optimized to give strong hybridization signals, even with low-copy mRN A molecules.

5

For a full discussion of each step of this protocol, along with more hints on enhancing the result of this often laborious and troublesome technique, see the previously published version of this report (Dijkman et al., 1995a; 1995b).

I. Probe selection Choosing the type of probe (i.e., DN A, RN A, or oligonucleotide) is essential for a good final result. For the optimization of the probe labeling, we have tested four methods of incorporating the DIG label: ! Direct labeling of cDN A molecules using

the DIG DN A Labeling Kit*.

! DIG labeling of oligonucleotides using

the DIG O ligonucleotide Tailing Kit*.

! DIG labeling of PCR products according

to the method of H annon et al. (1993).

! DIG labeling of cRN A molecules using

the DIG RN A Labeling Kit*. The RN A labeling method gave by far the best results. Therefore, we provide some hints on how to transcribe DIG-labeled cRN A molecules: ! For cRN A probe synthesis, subclone a

cDN A molecule in an appropriate vector. The vector should have sequences flanking the insert that allow the insert to be transcribed. Typically, commercially available vectors have SP6 and T7 RN A polymerase sites flanking the multiple cloning sites. ! Regulate the length of the cDN A molecule since this greatly affects the hybridization efficiency. Usually, a cDN A length between 200 and 500 nucleotides gives the best results, allowing efficient hybridization and good penetration of the tissue. ! When a molecule of interest is expressed as a high-copy RN A but the target RN A is masked by proteins, make probes between 100 and 200 nucleotides long for better penetration (Figure 1).

*Sold under the tradename of Genius in the US.

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a

II. Probe labeling " 1 After subcloning the cDN A molecule,

linearize the circular vector with restriction enzymes that cut the multiple cloning site in 2 orientations, allowing sense and antisense synthesis. C aution: C ontrol this lineariz ation step carefully by m onitoring the digestion w ith agarose gel electrophoresis. C ircular m olecules that are left in the digestion m ixture affect the transcription efficiency. 2 Extract the linearized molecules from the "

digestion mixture by phenol extraction.

" 3 Label the transcripts from the linearized

molecules with DIG-labeled nucleotides and the DIG RN A Labeling Kit* according to the instructions given in the kit, but with the following modifications: ! Perform the SP6 polymerase incubation at 40°C instead of 37°C. ! Precipitate the labeled molecules overnight at –20°C, rather than 30 min at –70°C. ! Monitor the transcription reaction by agarose gel electrophoresis to check for correct cRN A probe length. 4 Monitor probe labeling by spotting " diluted aliquots of the labeled cRN A probes on nylon membranes and analyzing with the DIG Luminescent Detection Kit*. See also Chapter 4, page 51. N ote: T he sense and antisense cR N A probes should be labeled equally as efficiently as the control D N A included in the D I G R N A L abeling Kit*.

b

c

5

5 If necessary, adjust the concentration of "

the labeled sense and antisense cRN A probes so they contain equal amounts of label. 6 Aliquot the cRN A probes in poly" propylene tubes at –70°C. N ote: R epeated freez ing and thaw ing affects the labeled cR N A probe.

#

Figure 1: RNA in situ hybridization on a frozen section of human psoriatic epidermis with DIGlabeled cRNA probes coding for human SKALP. Panel a: An antisense cRNA probe (150 bp) was used on a 10 µm thick section (magnificationx125). Panel b: An antisense cRNA probe (150 bp) was used on a 20 µm thick section (magnificationx500). Panel c: A sense cRNA probe (150 bp) was used on a 10 µm thick section (magnificationx270). Intense staining of both the stratum spinosum and stratum granulosum is observed with the antisense probe. *Sold under the tradename of Genius in the US.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to Tissues

III. Preparation of tissue sections

IV. Pretreatment of slides

N ote: For detection of low -copy R N A m olecules, alw ays prepare froz en sections, since paraffin em bedding causes a loss of approxim ately 30% of the R N A . T he m ethod described here has been optim iz ed for froz en sections.

C aution: Prepare all solutions for Procedures I V w ith w ater that has been treated w ith 0.1% D EPC .

" 1 For the processing of tissue, clean all

knives and other materials with RN ase ZAP (AMBIO N ) and work as aseptically as possible. 2 Remove the tissue from the animal, " immediately snap-freeze the tissue, and store it in liquid nitrogen. Work quickly to avoid degradation of RN A (Barton et al., 1993). " 3 Cut 10 µm thick frozen sections. N ote: To get a higher signal, cut sections thick er than 10 µm . For better signal localiz ation, cut sections thinner than 10 µm . 4 Mount tissue directly on Superfrost Plus "

slides (Menzel Gläser, O mnilabo, Breda, The N etherlands) to prevent detachment of the section during the RISH procedure.

" 1 Directly after mounting, heat the sections

on a stove for 2 min at 50°C to fix the RN A in the tissue. N ote: Vary the tim e (from 10–120 s) and tem perature (from 50°–90° C ) of the fixation step to accom m odate different types of tissue and R N A . 2 Dry the sections for 30 min. " " 3 Circle the sections with a silicone pen (DAKO A/S, Glostrup, Denmark) to prevent smudging of the substrate. 4 Use the following criteria to decide the " next step of the procedure: ! If the target tissue has lipid vesicles (Figure 2) that interfere with the RISH detection, go to Step 5. ! If the target tissue does not have lipid vesicles that interfere with the RISH detection, go to Step 6.



5



➞ Figure 2: RNA in situ hybridization on a frozen ! kidney section with a DIG-labeled cRNAprobe for mouse aminopeptidase A(without delipidization). The section was 10 µM thick and was taken from a male BALB/c mouse. Note the many nonspecific lipid vesicles in the section. The specific hybridization signal appears in cells of the glomerulus (arrows). Magnification, 600 x.

5 (O ptional) "

To minimize nonspecific background caused by lipid vesicles, do the following (all at room temperature): ! Delipidize the sections by extracting them for 5 min in chloroform. ! Dry the section to evaporate the chloroform. N ote: T his delipidation step m ay be om itted in pilot studies on a new tissue. 6 Fix the tissue sections as follows (all at " room temperature): ! Incubate tissue in PBS containing 4% paraformaldehyde for 7 min. ! Wash 1 x 3 min with PBS. ! Wash 2 x 5 min with 2 x SSC. N ote: 1 x SSC contains 150 m M N aC l, 15 m M sodium citrate; pH 7.2. 154

CONTENTS

INDEX

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to Tissues

V. Prehybridization, hybridization, and posthybridization

VI. Immunological detection

C aution: Prepare all solutions for Procedures V w ith w ater that has been treated w ith 0.1% D EPC .

temperature with 100 mM Tris-H Cl (pH 7.5), 150 mM N aCl. 2 Incubate the sections for 30 min at room " temperature with blocking buffer [100 mM Tris-H Cl (pH 7.5), 150 mM N aCl; saturated with blocking reagent]. " 3 Prepare a 1:200 dilution of alkaline phosphatase-conjugated anti-DIG antibody (polyclonal, Fab fragments, from sheep) in blocking buffer (from Step 2). 4 Incubate the sections for 120 min at room " temperature with the diluted anti-DIG antibody conjugate prepared in Step 3. 5 Wash the sections as follows: " ! 2 x 5 min at room temperature with 100 mM Tris-H Cl (pH 7.5), 150 mM N aCl. ! 1x10 min at room temperature with 100 mM detection buffer [Tris-H Cl (pH 9.5), 100 mM N aCl, 50 mM MgCl2]. 6 Cover the sections with detection buffer " containing 0.18 mg/ml BCIP, 0.34 mg/ml N BT, and 240 µg/ml levamisole. Incubate for 16 h at room temperature (for detection of low abundance RN A). N ote: T his dev elopm ent should be carried out w ith the slides standing up in a sm all container to prev ent nonspecifically conv erted substrate from falling onto the section.

" 1 Prehybridize each section for 60 min at

37°C in 100 µl hybridization buffer [4 x SSC ; 10% dextran sulfate; 1 x D enhardt’s solution (0.02% Ficoll® 400, 0.02% polyvinyl pyrolidone, 0.02% bovine serum albumin); 2 mM ED TA; 50% deionized formamide; 500 µg/ ml herring sperm D N A]. N ote: You m ay v ary the tem perature of the prehybridiz ation and hybridiz ation steps betw een 37° C and 50° C . 2 Perform hybridization as follows: " ! Remove and discard the buffer from

the prehybridization step.

! Cover each section with 100 µl hybridi-

zation buffer containing 200 ng/ml of DIG-labeled cRN A probe. C aution: D o not use cov erslips, since they decrease the signal up to fourfold. C aution: A probe concentration of 200 ng/ m l holds only if the cR N A w as labeled efficiently. I f, in Procedure I I , the cR N A probe w as not labeled as efficiently as the controls from the D I G R N A L abeling Kit*, adjust the concentration of the cR N A probe in the hybridiz ation m ixture. ! Incubate for 16 h at 37°C.

N ote: You m ay v ary the tem perature of the prehybridiz ation and hybridiz ation steps betw een 37° C and 50° C . " 3 After the hybridization, wash unbound

cRN A probe from the section as follows: ! 1 x 5 min with 2 x SSC at 37°C. N ote: To m ak e the w ash m ore stringent and w ash aw ay nonspecifically bound cR N A probe, low er the salt concentration or increase the form am ide concentration in the w ashing buffer and perform the w ashing step at a tem perature 5°C beneath the m elting tem perature of the probe.

" 1 Wash the sections for 5 min at room

5

" 7 Stop the color reaction by washing the

sections for 5 min in 10 mM Tris (pH 8), 1 mM EDTA.

VII. Counterstain " 1 Wash the sections for 5 min with distilled

H 2O at room temperature.

2 Counterstain the sections for 5–10 min "

with 1% methylene green at 37°C.

" 3 Repeat wash (from Step 1). 4 Mount coverslips in Kaiser’s solution "

(available from Merck).

! 3 x 5 min with 60% formamide in

0.2 x SSC at 37°C.

! 2 x 5 min with 2 x SSC at room tem-

perature.

*Sold under the tradename of Genius in the US.

CONTENTS

INDEX

155

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to Tissues

Results and discussion

Acknowledgments

Figure 3 shows a typical RISH signal obtained with this protocol and a labeled antisense cRN A probe coding for mouse aminopeptidase A. The protocol included the optional delipidation step with chloroform (Procedure IV, Step 5). Figure 4 shows the control reaction with the labeled sense cRN A probe.

The cDN A clone coding for the mouse aminopeptidase A cDN A sequence was kindly provided by Dr. Max D. Cooper from the University of Alabama at Birmingham, USA (Wu et al., 1990). The cRN A probe for elafin/SKALP was kindly provided by Dr. J. Schalkwijk, department of Dermatology, University H ospital N ijmegen, The N etherlands.

We have provided a standard protocol for RISH on low- and high-copy RN A molecules. Using this protocol, one should be able to perform RISH on each RN A molecule of interest, with only minor modifications depending on the abundance of the RN A of interest.







5 #

Figure 3: RNA in situ hybridization on a frozen kidney section with a DIG-labeled antisense cRNA probe for mouse aminopeptidase A (after delipidization). The section was 10 µm thick and was taken from a male BALB/c mouse. Pretreatment of the section with chloroform prior to the hybridization delipidized the section and reduced a nonspecific signal previously observed in lipid vesicles (Figure 2). The specific hybridization signal (arrows) was undiminished by the chloroform

156

#

Figure 4: RNA in situ hybridization on a frozen kidney section from the same mouse used in Figure 3 with a DIG-labeled sense cRNA probe for mouse aminopeptidase A. As in Figure 3, the section was 10 µm thick and was taken from a male BALB/c mouse. The section was pretreated with chloroform prior to the hybridization. No signal could be seen, indicating the specificity of the antisense hybridization signal seen in Figure 3. (magnification, 600x).

CONTENTS

INDEX

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to Tissues

References Alkemade, J. A. C.; Molhuizen, H. O. F.; Ponec, M.; Kempenaar, J. A.; Zeeuwen, P. L. J. M.; de Jongh, G. J.; van Vlijmen-Willems, I. M. J. J.; van Erp, P. E. J.; van de Kerkhof, P. C. M.; Schalkwijk, J. (1994) SKALP/elafin is an inducible proteinase inhibitor in human epidermal keratinocytes. J. Cell Sci. 107, 2335–2342. Assmann, K. J. M.; van Son, J. P. H. F.; Dijkman, H. B. P. M.; Koene, R. A. P. (1992) A nephritogenic rat monoclonal antibody to mouse aminopeptidase A. Induction of massive albuminuria after a single intravenous injection. J. Exp. Med. 175, 623–636. Dijkman, H. B. P. M.; Mentzel, S.; de Jong, A. S.; Assmann; K. J. M. (1995a) RNA In Situ hybridization using digoxigenin-labeled cRNA probes. Biochemica (U.S. version) No. 2 [1995], 23–27.

Dijkman, H. B. P. M.; Mentzel, S.; de Jong, A. S.; Assmann; K. J. M. (1995b) RNA In Situ hybridization using digoxigenin-labeled cRNA probes. Biochemica (worldwide version) No. 2 [1995], 21–25. Barton, A. J. L.; Pearson, R. C. A.; Najlerahim, A.; Harrison, P. J. (1993) Pre- and postmortem influences on brain RNA. J. Neurochem. 61, 1–11. Hannon, K.; Johnstone, E.; Craft, L. S.; Little, S. P.; Smith, C. K.; Heiman, M. L.; Santerre, R. F. (1993) Synthesis of PCR-derived, single-stranded DNA probes suitable for in situ hybridization. Anal. Biochem. 212, 421–427. Wu, Q.; Lahti, J. M.; Air, G. M.; Burrows, P. D.; Cooper, M. D. (1990) Molecular cloning of the murine BP-1/6C3 antigen: a member of the zinc-dependent metallopeptidase family. Proc. Natl. Acad. Sci. USA 87, 993–997.

Reagents available from Boehringer Mannheim for this procedure Reagent

Description

DIG RNA Labeling Kit* , **, ***

For RNA labeling with digoxigenin-UTP by in vitro transcription with SP6 and T7 RNA polymerases.

Cat. No.

Pack size

1175 025

1 Kit (2x10 labeling reactions)

EDTA

Powder

808 261 808 270 808 288

250 g 500 g 1 kg

DNA, from herring sperm

lyophilized sodium salt

223 646

1g

Blocking Reagent

For nucleic acid hybridization

1 096 176

50 g

Anti-Digoxigenin-AP

Anti-Digoxigenin, Fab fragments conjugated with alkaline phosphatase

1 093 274

150 U (200 µl)

Tris

For preparation of buffer solutions

127 434 708 968 708 976

100 g 500 g 1 kg

NBT solution

100 mg/ml nitroblue tetrazolium salt in 70% (v/v) dimethylformamide

1 383 213

3 ml (300 mg) (dilute prior to use)

BCIP solution

50 mg/ml 5-bromo-4-chloro-3-indolyl phosphate (BCIP), toluidinium salt in 100% dimethylformamide

1 383 221

3 ml (150 mg)

5

***Sold under the tradename of Genius in the US. ***EP Patent 0 324 474 granted to Boehringer Mannheim GmbH. ***Licensed by Institute Pasteur.

CONTENTS

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections W hole m ount I SH

Localization of the expression of the segmentation gene hunchback in Drosophila embryos with DIG-labeled DNA probes D r. D . Tautz , I nstitute for G enetics and M icrobiology, U niv ersity of M unich, G erm any.

I n situ hybridization to RN A on sections of early embryos provided one of the most significant advances for the analysis of spatially regulated transcripts of segmentation genes in D rosophila. It was therefore soon adapted to other systems. The procedure is, however, rather tedious and time-consuming. It depends on the quality of the sections and particularly on the quality of the probe. In contrast, antibody staining of the protein products from these genes in whole embryos simplified the procedure and allowed a much higher resolution. H owever, certain regulatory events have to be analyzed at the RN A level. Also antibody production itself is somewhat time consuming. We have therefore sought to develop an in situ hybridization technique which offers the same advantages as antibody staining. We found the use of digoxigenin-labeled probes to be highly successful for this purpose. These probes allow a resolution which is directly comparable to antibody staining in embryos, and both the sensitivity and the speed is superior to conventional radioactive methods.

5

The method is not limited to applications in Drosophila, but is apparently suitable for all kinds of tissues, at least for those in which antibody staining has been successful. We have already developed a protocol for the staining of specific cells in whole H ydra animals, which required only minor modifications (E. Kurz and C. David, University of Munich, Dept. of Zoology). The procedure given below has been published (Tautz and Pfeiffle, 1989).

158

I. Preparation of embryos N ote: A ll em bryos in this study cam e from w ild-type D rosophila flies and w ere collected on apple juice agar plates at interv als of 0–4 h at 25° C (W ieschaus and N üsslein-Volhard, 1986) ! 1 Collect the embryos in small baskets. 2 Wash embryos with water. ! ! 3 Dechorionate embryos for about 2–3 min

in a solution of 50% commercial bleach (about 5% sodium hypochlorite) in water. 4 Wash the embryos with 0.1% Triton ® X! 100. 5 Fix the embryos either with formald! ehyde (Procedure IIA) or paraformaldehyde (Procedure IIB). IIA. Formaldehyde fixation ! 1 Transfer each embryo into a glass

scintillation vial containing 4 ml of fixation buffer [0.1 M H epes, pH 6.9; 2 mM magnesium sulfate, 1 mM EGTA (from a 0.5 M stock EGTA solution that has been adjusted to pH 8 with N aO H )]. 2 Add to the scintillation vial 0.5 ml 37% ! formaldehyde solution and 5 ml heptane. ! 3 Shake the vial vigorously for 15–20 min to maintain an effective emulsion of the organic and the water phase. 4 Remove the lower phase and add 10 ml ! methanol. " If the embryos sink to the bottom at this stage, proceed to Step 5. " If the embryos do not sink to the bottom at this stage, remove the upper phase (heptane) and add more methanol. 5 Store the fixed embryos in methanol (up ! to several weeks) in the refrigerator.

CONTENTS

INDEX

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to Tissues

IIB. Paraformaldehyde fixation

Note: This protocol is more laborious, but leads, in some cases, to a lower background and to better preservation of the morphology. ! 1 Transfer

the embryos into a glass scintillation vial containing 1.6 ml of fixation buffer [100 mM H epes, pH 6.9; 2 mM magnesium sulfate, 1 mM EGTA (from a 500 mM stock EGTA solution that has been adjusted to pH 8 with N aO H )]. 2 Liquify a 20% stock solution of para! formaldehyde in water as follows: " Remove the stock 20% paraformaldehyde from its storage at –20°C. " H eat the stock to 65°C. " N eutralize with a little N aO H . ! 3 From the liquified stock 20% solution of paraformaldehyde, remove 0.4 ml and add it to the scintillation vial. 4 Also add 8 ml heptane to the vial. ! 5 Shake the vial and treat with methanol as ! in Steps 3 and 4 in Procedure IIA above. 6 Transfer each embryo to a 1.5 ml micro! centrifuge tube containing ME [90% methanol, 10% 0.5 M EGTA] . ! 7 Prepare PP solution (4% paraformaldehyde in PBS). N ote: PBS contains 130 m M N aC l and 10 m M sodium phosphate; pH 7.2. 8 Refix the embryos and dehydrate by !

passage through the following series of solutions containing ME and PP: " 5 min in ME:PP (7:3). " 5 min in ME:PP (1:1). " 5 min in ME:PP (3:7). " 20 min in PP alone. ! 9 Wash the embryos in PBS for 10 min and do either of the following: " Use the fixed embryos in Procedure III. or " If the fixed embryos are not to be used immediately, dehydrate them in a series of ethanol solutions (30% , 50% , then 70% ethanol) and store them at –20°C. C aution:D ehydrated, stored em bryos m ust be rehydrated before they can be used in Procedure I I I .

CONTENTS

INDEX

III. Pretreatment C aution: I n the steps below, use the norm al precautions for av oiding potential R N ase contam ination. ! 1 Treat the PBS for the following steps with

diethylpyrocarbonate (20 µl per 500 ml PBS), then autoclave the treated PBS. 2 Place each fixed embryo in a 1.5 ml ! microcentrifuge tube. Perform the incubations in Steps 3–8 below at room temperature on a revolving wheel. ! 3 Wash each embryo 3 x 5 min in 1 ml PBT (PBS plus 0.1% Tween ® 20). Note: Tween reduces non-specific adhesion of the embryos to the plastic surfaces. 4 Incubate the embryo for 3–5 min in 1 ml of !

PBS containing 50 µg/ml Proteinase K.

5 Stop the digestion by removing the !

Proteinase K solution, adding 1 ml PBT containing 2 mg/ml glycine to the embryo. Incubate for 2 min. C aution: T he proteinase digestion tim e is critical. I f it is too short, the back ground increases and sensitiv ity is lost. I f it is too long, the em bryos burst during the subsequent steps. 6 After the Proteinase K digestion, wash !

the embryo 2 x 5 min in 1 ml PBT.

! 7 Refix for 20 min in 1 ml PP (4%

paraformaldehyde in PBS).

5

8 Wash 3 x 10 min in 1 ml PBT. !

IV. Probe labeling The probes are DN A restriction fragments isolated from agarose gels. Label them according to the random priming method described in Chapter 4 of this manual.

159

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to Tissues

V. Hybridization

VI. Detection

! 1 Prepare the hybridization solution (H S)

! 1 Prepare a fresh dilution (1:2000 to

[50% formamide; 5 x SSC (where 1 x SSC contains 150 mM N aCl, 15 mM sodium citrate); 50 µg/ml heparin, 0.1% Tween ® 20; and 100 µg/ml sonicated and denatured salmon sperm DN A]. N ote: H S m ay be stored at –20° C .

1:5000) of alkaline phosphataseconjugated anti-DIG-antibody in PBT. N ote: T he antibody m ay be preabsorbed for 1 h w ith fixed em bryos to reduce the back ground.

2 Wash the embryos as follows: ! " 20 min in 1:1 H S:PBT. " 20–60 min in H S. ! 3 Prehybridize for 20–60 min in H S at

of 10 µg sonicated salmon sperm DN A. N ote: T he salm on sperm D N A should effectiv ely com pete out sim ple sequences and thus reduce the back ground.

diluted antibody conjugate for 1 h at room temperature on a revolving wheel. ! 3 Wash the embryo as follows: " 4x20 min in PBT. " 3x5 min in color buffer [100 mM N aCl, 50 mM MgCl2, 100 mM Tris (pH 9.5), 1 mM levamisole, 0.1% Tween ® 20]. N ote: L ev am isole is a potent inhibitor of lysosom al phosphatases.

5 Dilute the probe to approximately 0.5 µg !

4 Bring the embryos into a small dish with !

45°C in a water bath.

4 H eat denature the probe in the presence !

labeled probe per ml in H S.

6 Remove most of the prehybridization !

supernatant from the embryo, then add the heat-denatured probe and thoroughly mix. ! 7 H ybridize overnight at 45°C in a water bath without shaking. 8 Wash the embryos at room temperature ! in each of the following solutions: " 20 min in H S. " 20 min in 4:1 H S:PBT. " 20 min in 3:2 H S:PBT. " 20 min in 2:3 H S:PBT. " 20 min in 1:4 H S:PBT. " 2 x 20 min in PBT. N ote: U se a less extensiv e w ashing protocol if probe produces a low back ground.

5

2 Incubate each embryo with 500 µl freshly !

1 ml of color buffer. Add to this 4.5 µl N BT and 3.5 µl BCIP with thorough mixing. 4 Develop the color for 10–60 min in the ! dark. N ote: C olor dev elopm ent m ay occasionally be controlled under the binocular m icroscope and stopped in PBT before back ground dev elops. 6 Dehydrate the embryos in a series of !

ethanol solutions and mount in GMM (Lawrence et al., 1986).

Results Figure 1 shows typical results obtained with the above protocol. Panel a shows three embryos at different developmental stages which represent the dynamic expression pattern of hunchback. The embryo on the top right in Panel a shows the maternal gradient of hunchback RN A distribution; the embryo at the left the primary zygotic expression in two domains; and the middle embryo, the late zygotic expression. In Panel b, an enlarged section of an embryo shows the predominant cytoplasmic location of the hybridization signal. The section of the embryo corresponds to the frame depicted in Panel a although the photo was taken of a different embryo. Panel c shows an embryo after gastrulation, where individual cells of the developing nervous system express hunchback.

160

CONTENTS

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to Tissues

nuclei cytoplasm

yolk

b

a

c

#

References Lawrence, P. A.; Johnston, P.; Morata, G. (1986) Methods of marking cells. In: Roberts, D. B. (ed.) Drosophila, A Practical Approach. Oxford: IRL Press, pp. 229–242. Tautz, D.; Pfeiffle, C. (1989) A nonradioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals a translational control of the segmentation gene hunchback. Chromosoma (Berl.) 98, 81–85.

Wieschaus, E.; Nüsslein-Vollhard, C. (1986) Looking at embryos. In: Roberts, D. B. (ed.) Drosophila, A Practical Approach. Oxford: IRL Press, pp. 199–228.

Reagents available from Boehringer Mannheim for this procedure

Reagent

Description

Cat. No.

Pack size

Triton®

Vicous, liquid

789 704

100 ml

Hepes

Purity: 98% (from N)

223 778 737 151 242 608

100 g 500 g 1 kg

EGTA

Purity: > 98%

1 093 053

50 g

1 332 465

5x10 ml

161 519 745 723 1 000 144 1 092 766

25 mg 100 mg 500 mg 1g

DIG-High Prime* , **, *** Premixed solution for 40 random-primed DNA labeling reactions with DIG-11-dUTP, alkali-labile

1 585 606

160 µl (40 labeling reactions)

Anti-Digoxigenin-AP

750 units/ml Anti-Digoxigenin, Fab fragments conjugated to alkaline phosphatase

1 093 274

150 U (200 µl)

NBT solution

100 mg/ml nitroblue tetrazolium salt in 70% (v/v) dimethylformamide

1 383 213

3 ml (300 mg) (dilute prior to use)

BCIP solution

50 mg/ml 5-bromo-4-chloro-3-indolyl phosphate (BCIP), toluidinium salt in 100% dimethylformamide

1 383 221

3 ml (150 mg)

Tween®

X-100

20

Proteinase K

CONTENTS

Lyophilizate

INDEX

Figure 1: In situ hybridization on whole Drosophila embryos using a probe for the segmentation gene hunchback. Optical sections were obtained by appropriate focusing. For details of the panels, see text. Panel a (The bar at the top represents approx. 100 µm.) Panel b (The bar at the top represents approx. 5 µm.)

5

***EP Patent 0 324 474 granted to and EP Patent application 0 371 262 pending for Boehringer Mannheim GmbH. ***This product or the use of this product may be covered by one or more patents of Boehringer Mannheim GmbH, including the following: EP patent 0 649 909 (application pending). ***Licensed by Institute Pasteur.

161

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections W hole m ount I SH

Detection of even-skipped transcripts in Drosophila embryos with PCR/DIG-labeled DNA probes D r. N . Patel and D r. C . G oodm an, C arnegie I nstitute of Washington, Em bryology D epartm ent, Baltim ore, M aryland, U SA .

This protocol has been used to detect the transcript distribution of a number of genes by in situ hybridization, including evenskipped and seven-up, in whole mount Drosophila embryos, and engrailed Antennapedia in whole mount grasshopper embryos. The in situ hybridization and detection was performed essentially according to the protocol of Tautz and Pfeiffle (1989). This laboratory uses PCR rather than random primed labeling to prepare DIGlabeled probes because: " A much larger quantity of probe can be made with the same amount of starting nucleotide. " The ratio of labeled DN A to unlabeled starting material is much higher (especially important when transcripts to be detected are not very abundant). " PCR can produce strand-specific copies. Disadvantage: " It is more difficult to control the probe

size.

5

This laboratory has also found that biotin16-dUTP incorporated in the same way and detected with streptavidin-alkaline phosphatase is about three- to fivefold less sensitive than DIG-labeled probes in in situ hybridization experiments.

I. Probe labeling ! 1 Prepare the following stock solutions: " 10 x concentrated reaction mix:500 mM

KCl; 100 mM Tris-H Cl, pH 8.3; 15 mM MgCl2; 0.01% (w/v) gelatin. " 5 x concentrated dN TP mix: 1 mM dATP, 1 mM dCTP, 1 mM dGTP, 0.65 mM dTTP, 0.35 mM digoxigenin11-dUTP. " A primer stock solution containing either 30 ng/µl (approx. 5.3 mmol) primer 1 (e.g. generated by SP6 RN A polymerase) or 30 ng/µl (approx. 5.3 mmol) primer 2 (e.g. generated by T7 RN A polymerase). 2 With a restriction enzyme, linearize “dual ! promotor vector DN A” containing the insert, as one would to make run-off RN A transcripts. N ote: T he tw o different prim ers can be used to create an antisense strand and a sense strand (as control). ! 3 H eat inactivates the restriction enzyme. 4 Dilute the linearized DN A with water to !

a final concentration of about 100– 200 ng/µl. N ote: I f the insert is m uch ov er 3 k b, the probe produced w ill probably not represent the entire insert. 5 Set up the following reaction mix: ! " 9.25 µl water. " 2.5 µl 10 x concentrated reaction

mixture.

" 5.0 µl 5 x concentrated dN TP mix. " 5.0 µl primer 1 or 2 (from 30 ng/µl

stock).

" 2.0 µl linearized DN A (100–200 ng/µl).

6 Add 40 µl mineral oil, centrifuge, then !

boil the mix for 5 min.

! 7 To the mix, add 1.25 µl of 1 unit/µl Taq

DN A Polymerase (1.25 units of Taq Polymerase). [Final volume of reaction mix with Taq is 25 ml.] 8 Mix the contents of the reaction tube and ! then centrifuge for 2 min.

162

CONTENTS

INDEX

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections W hole m ount I SH

! 9 Incubate for 30 cycles in the PCR thermal

cycler under the following conditions: " Denaturation at 95°C for 45 s. " Annealing at 50°–55°C for 30 s. N ote: A nnealing tem perature depends on length of prim er. U se 55°C for a 21-m er. " Elongation at 72°C for 1.0–1.5 min. N ote: Elongation tim e depends on length of insert. U se 1 m in for 1.0 k b or less, 1.5 m in for 2.5 k b or m ore. 10 After the PCR run, add 75 µl distilled H 2O !

to the reaction tube, then centrifuge it.

11 Remove 90–95 µl of the reaction mix !

from beneath the oil.

! 12 To recover the DN A, do an ethanol

precipitation as follows: " Add N aCl to a final concentration of 0.1 M. " Add 10 µg of glycogen or tRN A as a carrier (0.5 µl of a 20 mg/ml stock). " Add 3 volumes of 100% EtO H . " Mix well and leave at –70°C for 30 min. " Centrifuge. " Wash pellet with 70% ethanol. " Dry under vacuum. O ptional: Perform a second ethanol precipitation as abov e. 13 Resuspend DN A pellet in 300 µl of !

II. Evaluation of labeling reaction ! 1 Prepare an aliquot of the probe as follows: " Remove 1 µl of probe from the

reaction mix.

" Add 5 µl of 5 x SSC. " Boil 5 min. " Q uick cool on ice. " Centrifuge.

2 Spot 1–2 µl of the probe aliquot onto a !

small nitrocellulose strip cut to fit into a 1.5 ml microcentrifuge tube or a 5 ml snap cap tube. ! 3 Bake the filter between two sheets of filter paper in an 80°C vacuum oven for 30 min. C aution: T he residual form am ide m ay cause the nitrocellulose to w arp. I f this is a problem , reduce the tim e in the bak ing ov en or do this spot test before the second precipitation (Procedure I , Step 12). N ote: U nincorporated nucleotide binds only slightly to the nitrocellulose. 4 Treat the baked filter as follows: ! " Wet the filter with 2 x SSC. " Wash filter 2 x 5 min in PBT (1 x PBS,

0.2% BSA, 0.1% Triton ® X-100).

" Place filter into a 1.5 ml micro-

centrifuge tube or 5 ml snap cap tube.

hybridization buffer [50% formamide; 5 x SSC (where 1 x SSC contains 150 mM N aCl, 15 mM sodium citrate); 50 µg/ml heparin, 0.1% Tween ® 20; and 100 µg/ ml sonicated and denatured salmon sperm DN A] (according to Tautz and Pfeifle, 1989). ! 14 To reduce the size of the single-stranded DN A, boil the probe for 40–60 min. N ote: For efficient penetration of and hybridiz ation to the em bryos, the av erage probe length should be about 50–200 bp.

5 Detect the labeled probe as follows: ! " Block the filter by incubating it

! 15 Dilute the probe as much as tenfold

described in the appropriate Boehringer Mannheim pack insert. ! 7 Stop the reaction when the spots are visible. N ote: Spots should be v isible w ithin a few m inutes and dark after 10–15 m in.

before use. N ote: O ptim al dilution v aries depending on the abundance of the transcript and back ground staining. We recom m end using the probe either undiluted (original 300 µl) or diluted up to threefold for the initial experim ent.

CONTENTS

INDEX

5

30 min in PBT.

" Incubate in PBT for 30–60 min with a

1:2000 dilution (in PBT) of alkaline phosphatase-conjugated anti-DIG antibody. " Wash 4 x 15 min in PBT. " Wash 2 x 5 min in a solution containing 100 mM N aCl; 50 mM MgCl2; 100 mM Tris, pH 9.5; 0.1% Tween ® 20. N ote: L ev am isole is not needed. 6 Develop color with N BT and BCIP as !

163

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III. Preparation of embryos, hybridization and detection Perform the preparation of the embryos, subsequent hybridization of the DIG-PCR probe, and immunological detection as described in the Tautz article, “Localization of the expression of the segmentation gene hunchback in Drosophila embryos with digoxigenin-labeled DN A probes,” page 158 in this manual.

Results a

b

#

Reference Tautz, D.; Pfeiffle, C. (1989) A nonradioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals a translational control of the segmentation gene hunchback. Chromosoma (Berl.) 98, 81–85.

5

Figure 1: Detection of the even-skipped phenotype on blastoderm stage Drosophila embryos. Panel a shows an in situ hybridization using the Drosophila even-skipped gene as probe. Panel b demonstrates the even-skipped product using the specific antibody as probe. The seven stripe pattern is associated with the even-skipped phenotype.

Reagents available from Boehringer Mannheim for this procedure Reagent

Description

PCR DIG Probe Synthesis Kit*

For generating highly sensitive probes labeled with DIG-dUTP (alkali-labile) in the polymerase chain reaction (PCR) using a ratio of 1+2 (DIG-dUTP:dTTP)

Tween® 20

*This product is sold under licensing arrangements with Roche Molecular Systems and The PerkinElmer Corporation. Purchase of this product is accompanied by a license to use it in the Polymerase Chain Reaction (PCR) process in conjunction with an Authorized Thermal Cycler. For complete license disclaimer, see inside back

164

Cat. No.

Pack size

1 636 090

1 Kit (25 reactions)

1 332 465

5x10 ml

Glycogen

From rabbit liver

106 089

500 mg

BSA

Highest quality, lyophilizate

238 031 238 040

1g 10 g

Triton® X-100

Vicous, liquid

789 704

100 ml

Anti-Digoxigenin-AP

750 units/ml Anti-Digoxigenin, Fab fragments conjugated to alkaline phosphatase

1 093 274

150 U (200 µl)

NBT solution

100 mg/ml nitroblue tetrazolium salt in 70% (v/v) dimethylformamide

1 383 213

3 ml (300 mg) (dilute prior to use)

BCIP solution

50 mg/ml 5-bromo-4-chloro-3-indolyl phosphate (BCIP), toluidinium salt in 100% dimethylformamide

1 383 221

3 ml (150 mg)

CONTENTS

INDEX

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections W hole m ount I SH

Wholemount fluorescence insitu hybridization(FISH) of repetitive DNA sequences on interphase nuclei of the small cruciferous plant Arabidopsis thaliana Serge Bauw ens1 and Patrick Van O ostv eldt 2 L aboratory for G enetics, G ent U niv ersity, G ent, Belgium 2 L aboratory for Biochem istry and M olecular C ytology, G ent U niv ersity, G ent, Belgium

1

H ybridizing fluorescently labeled DN A probes in situ to the chromatin of interphase nuclei in whole mounts allows the study of nuclear architecture in morphologically well preserved specimens. Fluorescent labeling of the DN A probes (either directly or indirectly) allows the simultaneous, but differential, detection of several sequences (e.g. Lengauer et al., 1993; N ederlof et al., 1990). Also, fluorescent signals can be imaged by confocal microscopy so that stacks of optical section images can be recorded through the whole mounts. Multiple labeling FISH on whole mounts therefore allows the study of the relative positions of different chromosomes or chromosome segments in individual interphase nuclei as well as between nuclei of related cells. In the experiments presented here, two tandemly repeated sequences, rDN A and a 500 bp repeat sequence, were hybridized to interphase nuclei of seedlings and flowers (inflorescences) of the small cruciferous plant Arabidopsis thaliana. The procedure used in the experiments, based on the protocols published by Ludevid et al. (1992) and Tautz and Pfeifle (1989), was described earlier by Bauwens et al. (1994).

I. Seed sterilization and germination N ote: Procedure I is based on the protocol of Valv ek ens et al. (1988). ! 1 Surface sterilize seeds of Arabidopsis

thaliana (C24) by immersing them as follows: " 2 min in 70% (v/v) ethanol. " 15 min in a solution of 5% (v/v) N aO Cl and 0.05% (v/v) Tween ® 20. 2 Wash seeds 5 times in sterile, distilled ! water.

CONTENTS

INDEX

! 3 Pipette onto germination medium (1x

Murashige and Skoog salt mixture (Flow Laboratories, USA); 0.5 g/L 2-(N morpholino)ethane sulphonic acid (MES), pH 5.7 (adjusted with 1 M KO H ); 0.8% (w/v) Bacto-agar (Difco Laboratories, USA). 4 Allow the seeds to germinate at room ! conditions for 4 days. 5 Transfer part of the seedlings to soil. ! 6 Grow plants under continuous light ! conditions at desk temperature. ! 7 H arvest flowers and inflorescences after 3–4 weeks.

II. Tissue fixation ! 1 Place approximately 30–40 seedlings or a

few flowers or inflorescences in a glass vial containing: 4.365 ml fixation buffer [1.1 x PBS; 0.067 M EGTA, pH 7.5 (adjusted with N aO H )]. N ote: 10 x PBS contains 1.3 M N aC l, 0.0027 M KC l, 0.07 M N a2H PO 4, 0.03 M N aH 2PO 4; pH 7.2. " 0.135 ml 37% formaldehyde (Sigma, USA). " 0.5 ml DMSO . N ote: Final concentrations in v ial are 1% form aldehyde and 10% D M SO .

5

2 Rock the glass vial for 25 min at room !

temperature.

! 3 Remove the fixative and rinse as follows: " 2 x with 5 ml methanol. " 4 x with 5 ml ethanol. 4 Discard the last ethanol wash, then cover !

sample with a final 5 ml ethanol.

5 Leave sample at –20°C for 2–4 days. !

C aution: Keeping the m aterial for longer periods of tim e in ethanol m ak es it brittle.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections W hole m ount I SH

III. Labeling of the probe DNA

IV. Pretreatment

! 1 Use the following as hybridization

! 1 Remove ethanol from seedlings or flow-

probes: " A mixture of three ribosomal DN A (rDN A) inserts, each cloned into pBS (I)KS+ (Stratagene, USA) from A. thaliana. The inserts contain the 5.8S, 18S and 25S rRN A genes, as well as the intergenic region (IGR) (Unfried and Gruendler, 1990; Unfried et al., 1989). " A 500 bp repeat DN A sequence, cloned into pGem-2 (Promega, USA). The repeat is one of three classes of highly repetitive, tandemly arranged DN A sequences in A. thaliana (Simoens et al., 1988). 2 Label undigested samples of both probes ! according to the nick translation procedures in Chapter IV of this manual. Use the following labels: " Label the rDN A with either DIGdUTP or fluorescein-dUTP. N ote: T he concentration of substituted nucleotide in the nick translation labeling m ixture should be the sam e for either fluorescein-dU T P or D I G -dU T P.

ers (or inflorescences) and transfer material to microcentrifuge tubes. 2 Fix each sample as follows: ! " Rinse 2 x with 1 ml ethanol. " Replace ethanol with 1 ml ethanol/ xylene (1:1) and incubate for 30 min. " Rinse 2 x with 1 ml ethanol. " Rinse 2 x with 1 ml methanol. " Replace methanol with 1 ml of a 1:1 mixture of methanol and [PBT containing 1% (v/v) formaldehyde]. Rock for 5 min. N ote: PBT contains 1 x PBS and 0.1% (v / v ) Tw een ® 20. ! 3 Post-fix sample for 25 min in 1 ml PBT containing 1% formaldehyde. 4 Remove fixative and rinse sample with ! 5 x1 ml PBT. 5 Wash sample 3 x 1 ml of 2 x SSC (each ! wash, 5 min). N ote: 1 x SSC contains 150 m M N aC l and 15 m M sodium citrate, pH 7.0.

" Label the 500 bp repeat DN A with

DIG-dUTP.

5

166

6 Digest with RN ase A (100 µg/ml in !

2 x SSC) for 1 h at 37°C.

! 7 Wash 3 x 5 min with 1 ml PBT. 8 Digest with Proteinase K (40 µg/ml in !

PBT) for 8 min at 37°C.

! 3 After nick translation, treat each labeled

! 9 After the Proteinase K digestion, do the

probe as follows: " Co-precipitate 1 µg of labeled DN A with 55 µg of sonicated salmon sperm DN A (Sigma, USA). " Redissolve probe in 25 µl H 2O to a concentration of 40 ng labeled DN A per µl.

following: " Rinse 2 x with 1 ml PBT. " Wash 2 x 2 min with 1 ml PBT. " Rinse 2 x with 1 ml PBT. 10 Postfix a second time for 25 min with ! 1 ml PBT containing 1% formaldehyde. 11 Remove fixative and rinse 5 x with 1 ml ! PBT.

CONTENTS

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections W hole m ount I SH

V. In situ hybridization ! 1 Wash each sample 10 min with 1 ml of a

1:1 mixture of PBT and hybridization solution [hybridization solution contains 50% formamide (Ultra Pure from USB, USA) in 2 x SSC]. 2 Rinse sample with 2 x1 ml hybridization ! solution. ! 3 Remove the hybridization solution and add the following to each sample (to produce a final volume of 500 µl of hybridization solution): " 250 µl formamide. " 50 µl 20 x SSC. " Enough H 2O to make a total volume, including probes, of 500 µl. " 25 µl of each labeled rDN A probe (for single or double labeling experiments). " 25 µl labeled 500 bp repeat probe (for double labeling experiments only). N ote: Each labeled probe has a final concentration of 2 ng/ µl. N ote: T his incubation m ixture can be used for either a single label hybridiz ation (fluorescein-labeled probe) w ith direct detection; a single label hybridiz ation (D I G -labeled probe) w ith indirect detection; or a double label hybridiz ation (both fluorescein- and digoxigeninlabeled probes). See Procedure VIII below for details on these different types of experiments. 4 Treat the sample as follows: ! " Denature target and probe in hybridi-

zation solution for 4 min at 100°C.

" Place immediately on ice for 3 min. " Centrifuge very briefly. " Incubate overnight at 37°C to hybridize

probe and target. C aution: I f using a directly labeled (i.e., fluorescent) probe, perform the hybridiz ation incubation and the rest of the procedure in the dark .

CONTENTS

INDEX

VI. Pre-absorption of antibodies (for indirect detection only) ! 1 Prepare powdered A. thaliana root or

seedling extract as follows: " Grind the root or seedling under liquid nitrogen. " Extract the ground powder with acetone under liquid nitrogen. " Decant the acetone supernatant. " Let the residual acetone evaporate from the precipitate. " Use the dry, powdered precipitate in the pre-absorption procedure below. 2 Dilute each detection antibody, in 4 x SSC ! containing 1% (w/v) BSA, to the working dilution suggested by the manufacturers and a final volume of 500 µl. ! 3 Add approximately 2 mg of powdered A. thaliana root or seedling extract (from Step 1 above) to the diluted antibody. 4 Pre-absorb the antibodies overnight at ! 15°C, in the dark. 5 Centrifuge the pre-absorption mixture. ! 6 Use the supernatant in the immunocyto! chemical detection reaction.

VII. Posthybridization washes ! 1 After overnight hybridization at 37°C

(Procedure V, Step 4), treat the hybridization sample as follows: " Remove the hybridization solution. " Wash the sample for 1 h in fresh hybridization solution at 37°C. " Wash the sample 4 x 30 min in hybridization solution at 37°C. " If performing an in situ hybridization experiment with directly labeled probe DN A (rDN A) in a single labeling experiment, proceed to procedure VIII a. " If performing an in situ hybridization experiment requiring indirect detection with antibodies, proceed to step 2. 2 Bring the seedlings or flowers (or inflo! rescences) gradually to 4 x SSC through the following washes (all at room temperature): " 20 min with a 3:1 mix of hybridization solution and 4 x SSC. " 20 min with a 1:1 mix of hybridization solution and 4 x SSC. " 20 min with a 1:3 mix of hybridization solution and 4 x SSC. " 4 x 5 min with 4 x SSC.

5

167

Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections W hole m ount I SH

VIII. Immunocytochemical detection Use the immunocytochemical detection schemes detailed in Table 1 to analyze the single and double labeling in situ hybridization experiments.

Type of Experiment

Probe

Label

Single label, direct detection

rDN A

FluoresceindUTP

Single label, indirect detection

rDN A

DIG-dUTP

Double label

rDN A

FluoresceindUTP DIG-dUTP

500 bp

Antibody a 1st (detecting) 2nd (amplifying) – Fluorescein-conjugated anti-DIG antibody (from sheep)b



VIIIa

Fluorescein-conjugated anti-sheep antibody (from donkey)c

– Tetramethylrhodamine-conjugated anti-DIG antibody (from sheep)d

Procedure to Follow



VIIIb

VIIIb

Tetramethylrhodamine-conjugated anti-sheep antibody body (from rabbit)e

#

Table 1: Immunocytochemical detection schemes for single and double labeling experiments

5

a All antibodies should be preabsorbed, according to Procedure VI above. b Fab fragments. c Fab fragments, from Sigma, USA. d Fab fragments. e Whole molecule, from Chemicon, USA.

168

VIIIa. Direct detection ! 1 Bring the samples to 1 x PBS gradually

through the following washes (all at room temperature): " 20 min with a 3:1 mix of hybridization solution and 1 x PBS. " 20 min with a 1:1 mix of hybridization solution and 1 x PBS. " 20 min with a 1:3 mix of hybridization solution and 1 x PBS. " 4 x 15 min in 1 ml 1 x PBS. 2 Proceed to the staining and mounting ! procedure (Procedure IX).

VIIIb. Indirect detection ! 1 After the posthybridization washes (Pro-

cedure VII), remove the 4 x SSC solution from the samples. 2 Add the first pre-absorbed antibody ! (from Table 1) to the sample and incubate overnight at 15°C in the dark. ! 3 Remove the first antibody and wash the material 4 x15 min in 1 ml 4 x SSC containing 0.05% (v/v) Tween ® 20. 4 Depending on whether you are using an ! amplifying (second antibody), do either of the following: " If you use an amplifying antibody, proceed to Step 5. " If you do not use an amplifying antibody, proceed to Step 7. 5 If amplifying the signal, wash the sample ! 4 x 15 min with 4 x SSC containing 1% bovine serum albumin. 6 Remove the wash solution; add the ! second pre-absorbed antibody (from Table 1) to the sample, and incubate overnight at 15°C in the dark. ! 7 After the last (first or second) antibody incubation, remove the antibody and wash the sample 4 x 15 min in 1 ml 1 x PBS. 8 Proceed to the staining and mounting ! procedure (Procedure IX).

CONTENTS

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections W hole m ount I SH

IX. Staining and mounting

Xb. Confocal fluorescence microscopy

! 1 Counterstain the sample as follows: " For single labeling experiments with

images with the MRC-600 Confocal Scanning Laser Microscope (CSLM) System (Bio-Rad, USA). Attach the CSLM to the DIAPH O T 300 inverted microscope fitted with appropriate lenses (same microscope and lenses as described in Procedure Xa, Step 1 above). 2 Use the K1/K2 filter block combinations ! (Bio-Rad, USA) and either: " The 568 nm line from a KryptonArgon laser (Ion Laser Technology, Utah, USA), to image the propidium iodide-stained interphase nuclei and the tetramethylrhodamine-labeled hybrids. " The 488 nm line from the same Krypton-Argon laser, to image the fluorescein-labeled hybrids.

fluorescein-conjugated antibodies or nucleotides: Counterstain the chromatin of the interphase nuclei with 0.5 µg/ml propidium iodide (in 1 x PBS) for 1 h in the dark. " For double labeling experiments with fluorescein- and tetramethylrhodamineconjugated nucleotides and antibodies: Counterstain the chromatin of the interphase nuclei with 0.2 µg/ml DAPI (in 1 x PBS) for 1 h in the dark. N ote: D A PI is 4,6'-diam idino-2phenylindole. 2 Place a few seedlings or flowers (or part ! of an inflorescence) on a slide. ! 3 Apply some tape to the slides to create a support for the cover slip, so as not to crush the seedlings or flowers. 4 Apply a drop of antifade reagent ! (Vectashield, Vector Laboratories, USA) and cover with a cover slip.

X. Fluorescence microscopy Xa. Conventional fluorescence microscopy ! 1 Visually

inspect the slides on a DIAPH O T 300 inverted microscope (N ikon, Japan), fitted with either a 60 x, N A 1.40 oil immersion lens (O lympus, Japan) or an N PL FLUO TAR, 40 x, N A 1.30 oil immersion lens (Leitz, FRG). 2 Use the following filter combinations ! (Chroma Technology Corp., USA): " To localize propidium iodide-stained nuclei and tetramethylrhodaminelabeled hybrids: filter block 31014 404. " To localize DAPI-stained nuclei: filter block 31000 404. " To localize fluorescein-labeled hybrids: filter block 31 001 404. ! 3 As light source, use a mercury arc lamp (100 W).

! 1 Record

Results and discussion Figures 1 and 2 show some of the results obtained by FISH of rDN A and the 500 bp repeat on whole mounts of flowers and seedlings from A. thaliana. The rDN A from A. thaliana covers about 5.7 Mbp per haploid genome (Meyerowitz and Pruitt, 1985), and is distributed over two large tandem repeats on chromosomes 2 and 4 (Maluszynska and H eslop-H arrison, 1991; Murata et al. 1990). Diploid interphase nuclei from A. thaliana exhibit, however, a number of rDN A-loci ranging from two to more than four (Bauwens et al. 1991) as can clearly be seen from the merged image in Figure 1.

5

The 500 bp repeat sequence covers about 0.3–0.6 Mbp per haploid genome (Bauwens et al., 1991; Simoens et al., 1988) and exhibits a chromosome-specific large cluster (Bauwens and Van O ostveldt, 1991; Bauwens et al., 1991), resulting in two distinct signals in diploid interphase nuclei as can be seen from the merged image in Figure 2. Some helpful remarks should be made about confocal observation of fluorescent signals in whole mounts.

CONTENTS

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections W hole m ount I SH

Sequential excitation with the 568 nm and the 488 nm lines of the Kr-Ar-laser and the K1/K2 filter combinations from the BioRad MRC-600 CSLM, allowed a clear separation of the red (tetramethylrhodamine) and the green (fluorescein) signal. [See the signals from the 500 bp repeat (red) and the rDN A (green) probes in Figure 2.]. Especially, the yellow 568 nm line allows specific excitation of the red fluorescing dyes such as tetramethylrhodamine or, preferably, Texas Red™ with almost no excitation of fluorescein.

#

Figure 1: Whole mount FISH with rDNA on a developing flower from A. thaliana. The image is a merged image representing part of the pistil of a developing flower, showing fluorescein-labeled rDNA loci (yellow-green) and propidium iodidestained interphase nuclei (red). An extended focus image of 15 optical sections through the pistil was created by maximum brightness projection before merging the ‘green’ (rDNA) and ‘red’ (nuclei) images. Bar 25 µm ^= 17 mm.

Confocal observation of the preparations appeared to be absolutely necessary to obtain clear images of the signals, especially in the very dense meristematic tissues of the seedling roottips and the developing flowers in the inflorescences. This was especially true for the weaker signal of the 500 bp repeat that, in many cases, could not be observed through the autofluorescence haze by conventional fluorescence microscopy. Acquiring digital images through confocal microscopy has the added advantage that images recorded at different wavelengths can be easily and accurately merged and compared. Finally, the development of FISH protocols on whole mounts for localizing mRN A sequences, as already suggested by de Almeida Engler et al. (1994), will make it possible to follow the expression of different genes simultaneously at the cellular level, in three dimensions, through confocal observation.

5

References #

Figure 2: Whole mount double labeling FISH with rDNA and the 500 bp repeat on a seedling from A. thaliana. This merged image represents part of the meristematic zone of a seedling roottip. It shows fluorescein-labeled rDNA loci (green) and tetramethylrhodamine-labeled 500 bp repeat loci (red). An extended focus image of 20 optical sections through the meristematic zone of the roottip was created by maximum brightness projection before merging the ‘green’ (rDNA) and ‘red’ (500 bp repeat) images. Bar 25 µm ^= 15 mm.

Bauwens, S.; Katsanis, K.; Van Montagu, M.; Van Oostveldt, P.; Engler, G. (1994) Procedure for whole mount fluorescence in situ hybridization of interphase nuclei on Arabidopsis thaliana. Plant J. 6, 123–131. Bauwens, S.; Van Oostveldt, P. (1991) Cytogenetic analysis on interphase nuclei with non-isotopic in situ hybridization and confocal microscopy. Med. Fac. Landbouww. Rijksuniv. Gent. 56/3a, 753–758. Bauwens, S.; Van Oostveldt, P.; Engler, G.; Van Montagu, M. (1991) Distribution of the rDNA and three classes of highly repetitive DNA in the chromatin of interphase nuclei of Arabidopsis thaliana. Chromosoma 101, 41–48. de Almeida Engler, J.; Van Montagu, M.; Engler, G. (1994) Hybridization in situ of whole-mount messenger RNA in plants. Plant Mol. Biol. Reporter 12, 321–331.

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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections W hole m ount I SH

Lengauer, C.; Speicher, M. R.; Popp, S.; Jauch, A.; Taniwaki, M.; Nagaraja, R.; Riethman, H. C.; Donis-Keller, H.; D’Urso, M.; Schlessinger, D.; Cremer, T. (1993) Chromosomal bar codes produced by multicolor fluorescence in situ hybridization with multiple YAC clones and whole chromosome painting probes. Hum. Mol. Gen. 2, 505–512. Ludevid, D.; Höfte, H.; Himelblau, E.; Chrispeels, M. J. (1992) The expression pattern of the tonoplast intrinsic protein !-TIP in Arabidopsis thaliana is correlated with cell enlargement. Plant Physiol. 100, 1633–1639. Maluszynska, J.; Heslop-Harrison, J. S. (1991) Localization of tandemly repeated DNA sequences in Arabidopsis thaliana. Plant J. 1, 159–166. Meyerowitz, E. M.; Pruitt, R. E. (1985) Arabidopsis thaliana and plant molecular genetics. Science 229, 1214–1218. Murata, M.; Varga, F.; Maluszynska, J.; Gruendler, P.; Schweitzer, D. (1990) Chromosomal localization of the ribosomal RNA genes in Arabidopsis thaliana by in situ hybridization. In: Schweitzer, D.; Peuker, K.; Loidl, J. (Eds) Fourth International Conference on Arabidopsis Research, Vienna, Abstracts, 5.

Nederlof, P. M.; van der Ploeg, S.; Wiegant, J.; Raap, A. K.; Tanke, H. J.; Ploem, J. S.; van der Ploeg, M. (1990) Multiple fluorescence in situ hybridization. Cytometry 11, 126–131. Simoens, C. R.; Gielen, J.; Van Montagu, M.; Inzé, D. (1988) Characterization of highly repetitive sequences of Arabidopsis thaliana. Nucleic Acids Res. 16, 6753–6766. Tautz, D.; Pfeifle, C. (1989) A nonradioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98, 81–85. Unfried, I.; Gruendler, P. (1990) Nucleotide sequence of the 5.8S and 25S rRNA genes and of the internal transcribed spacers from Arabidopsis thaliana. Nucleic Acids Res. 18, 4011. Unfried, I.; Stocker, U.; Gruendler, P. (1989) Nucleotide sequence of the 18S rRNA gene from Arabidopsis thaliana Co 10. Nucleic Acids Res 7, 7513. Valvekens, D.; Van Lijsebettens, M.; Van Montagu, M. (1988) Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection. Proc. Natl. Acad. Sci. USA 85, 5536–5540.

Reagents available from Boehringer Mannheim for this procedure Reagent

Description

Cat. No.

Pack size

DIG-Nick Translation Mix*

For generation of highly sensitive probes for 1745 816 in situ hybridization with digoxigenin-11-dUTP. Premixed solution for 40 labeling reactions

160 µl

Nick Translation Mix*

For generation of highly sensitive probes for 1745 808 fluorescence in situ hybridization. The Nick Translation Mix for in situ probes is designed for direct fluorophore-labeling of in situ probes.

200 µl

Fluorescein-12-dUTP

Tetralithium salt, solution, 1 mmol/l

1 373 242

25 nmol (25 µl)

dNTP Set

Set of dATP, dCTP, dGTP, dTTP, lithium salts, solutions

1 277 049

1 set, 4x10 µmol (100 µl)

Anti-DigoxigeninRhodamine

Fab Fragments from sheep

1 207 750

200 µg

Anti-DigoxigeninFluorescein

Fab Fragments from sheep

1 207 741

200 µg

1 332 465

5x10 ml

Tween® 20 EGTA

Purity: > 98%

1 093 053

50 g

Proteinase K

Lyophilizate

161 519 745 723 1 000 144 1 092 766

25 mg 100 mg 500 mg 1g

RNase A

Dry powder

109 142 109 169

25 mg 100 mg

5

*This product or the use of this product may be covered by one or more patents of Boehringer Mannheim GmbH, including the following: EP patent 0 649 909 (application pending).

CONTENTS

INDEX

171

References

CONTENTS

INDEX

R eferences

In this chapter general references of publications on the use of digoxigenin in in situ hybridization experiments are listed alphabetically to give to the reader an overview on the wide range of applications presently available.

1. mRNA target A. DNA probe a. RPL Aida, T.; Yamada, H.; Asano, G. (1990) Expression of type VI procollagen and prolyl-4-hydroxylase mRNA in carbon tetra-chloride induced liver fibrosis studied by in situ hybridization. Acta Histochem. Cytochem. 23 (6), 817–824. Ayoubi, T. A. Y.; van Duijnhoven, H. L. P.; Coenen, A. J. M.; Jenks, B. G.; Roubos, E. W.; Martens, G. J. M. (1991) Coordinated expression of 7B2 and ! MSH in the melanotrope cells of Xenopus laevis. Cell Tissue Res. 264, 329–334. Bier, E.; Jan, L. Y.; Nung Jan, Y. (1990) Rhomboid, a gene required for dorsoventral axis establishment and peripheral nervous system development in Drosophila melanogaster. Genes & Development 4, 190–203.

6

Corbin, V.; Michelson, A. M.; Abmayr, S. M.; Neel, V.; Alcamo, E.; Maniatis, T.; Young, M. W. (1991) A role for the drosophila neurogenic genes in mesoderm differentiation. Cell Vol. 67, 311–323. Crespo, P.; Angeles-Ros, M.; Ordovas, J. M.; Rodriguez, J. C.; Ortiz, J. M.; Leon, J. (1992) Foam cells from aorta and spleen overexpress apolipoprotein E in the absence of hypercholesterolemia. Biochem. & Biophys. Res. Communications 183 (2), 514–523.

Bland, Y. S.; Critchlow, M. A.; Ashhurst, D. E. (1991) Digoxigenin as a probe label for in situ hybridization on skeletal tissue. Histochem. J. 23, 415–418.

Crowley, T. E.; Hoey, T.; Liu, J.-K.; Jan, Y. N.; Jan, L. Y.; Tjian, R. (1993) A new factor related to TATA-binding protein has highly restricted expression patterns in Drosophila. Nature 361, 557–561.

Blochlinger, K.; Bodmer, R.; Jan, L. Y.; Jan, Y. N. (1990) Patterns of expression of cut, a protein required for external sensory organ development in wild-type and cut mutant Drosophila embryos. Genes & Development 8, 1322–1331.

Donly, B. C.; Ding, Q.; Tobe, S. S.; Bendena, W. G. (1993) Molecular cloning of the gene for the allatostatin family of neuropeptides from the cockroach Diploptera punctata. Proc. Natl. Acad. Sci. USA 90, 8807–8811.

Bochenek, B.; Hirsch, A. (1990) In situ hybridization of nodulin mRNAs in root nodules using nonradioactive probes. Plant Mol. Biol. Reporter 8 (4), 237–248.

Doria-Medina, C. L.; Lund, D. D.; Pasley, A.; Sandra, A.; Sivitz, W .I. (1993) Immunolocalization of GLUT-1 glucose transporter in rat skeletalmuscle and in normal and hypoxic cardia tissue. Am. J. Physiol. 265, E454–E464.

Breitschopf, H.; Suchanek, G.; Gould, R. M.; Colman, D. R.; Lassmann, H. (1992) In situ hybridization with digoxigenin-labeled probes: sensitive and reliable detection method applied to myelinating rat brain. Acta Neuropathol. 84, 581–587. Brown, D. W.; Welsh, R. M.; Like, A. A. (1993) Infection of peripancreatic lymph nodes but not islets precedes kilham rat virus-induced diabetes in BB/Wor rats. J. Virol. 67 (10), 5873–5878. Casanova, J.; Struhl, G. (1993) The torso receptor localizes as well as transduces the spatial signal specifying terminal body pattern in Drosophila. Nature 362, 152–155. Cohen, B.; Wimmer, E. A.; Cohen, S. M. (1991) Early development of leg and wing primordia in the Drosophila embryo. Mechan. Develop. 33, 229–240.

174

Cohen, S. M. (1990) Specification of limb development in the drosophila embryo by positional cues from segmentation genes. Nature 343, 173–177.

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CONTENTS

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Fukuda, T.; Nakajima, H.; Fukushima, Y.; Akutsu, I.; Numao, T.; Majima, K.; Motojima, S.; Sato, Y.; Takatsu, K.; Makino, S. (1994) Detection of interleukin-5 messenger RNA and interleukin-5 protein in bronchial biopsies from asthma by nonradioactive in situ hybridization and immunohistochemistry. J. Allergy Clin. Immunol. 94, 584–593. Grabner, M.; Bachmann, A.; Rosenthal, F.; Striessnig, J.; Schulz, C.; Tautz, D.; Glossmann, H. (1994) Insect calcium channels. Molecular cloning of an !1-subunit from housefly (Musca domestica) muscle. FEBS Letters 339, 189–194. Haenlin, M.; Kramatschek, B.; Campos-Ortega, J. A. (1990) The pattern of transcription of the neurogenic gene delta of Drosophila melanogaster. Develop. 110, 905–914. Heemskerk, J.; DiNardo, S.; Kostriken, R.; O’Farell, P. H. (1991) Multiple modes of engrailed regulation in the progression towards cell fate determination. Nature 352, 404–410. Hülskamp, M.; Pfeifle, C.; Tautz, D. (1990) A morphogenetic gradient of hunchback protein organizes the expression of the gap genes krüppel and knirps in the early Drosophila embryo. Nature 346, 577–580. Hukkanen, V.; Heino, P.; Sears, A. E.; Roizman, B. (1990) Detection of Herpes Simplex Virus latencyassociated RNA in mouse trigeminal ganglia by in situ hybridization using nonradioactive digoxigeninlabeled DNA and RNA probes. Methods Mol. Cell. Biol. 2, 70–81. Kellerman, K. A.; Mattson, D. M.; Duncan, I. (1990) Mutations affecting the stability of the fushi tarazu protein of Drosophila. Genes & Development 4, 1936–1950. Krauss, S.; Johansen, T.; Korzh, V.; Fjose, A. (1991) Expression pattern of zebrafish pax genes suggest a role in early brain regionalization. Nature 353, 267–270. Kumar, G.; Patel, D.; Naz, R. K. (1993) c- MYC mRNA is present in human sperm cells. Cell. & Mol. Biol. Res. 39, 111–117. Levine, P. H.; Jahan, N.; Murari, P.; Manak, M.; Jaffe, E. S. (1992) Detection of human herpesvirus 6 in tissues involved by sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman Disease). J. Infect. Dis. 166, 291–295. Maggiano, N.; Larocca, L. M.; Piantelli, M.; Ranelletti, F. O.; Lauriola, L.; Ricci, R.; Capelli, A. (1991) Detection of mRNA and hnRNA using a digoxigenin labeled cDNA probe by in situ hybridization on frozen tissue sections. Histochem. J. 23, 69–74. Masucci, J. D.; Miltenberger, R. J.; Hoffman, F. M. (1990) Pattern-specific expression of the Drosophila decapentaplegic gene in imaginal disks is regulated by 3’cisregulatory elements. Genes & Development 4, 2011–2023.

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Mlodzik, M.; Baker, N. E.; Rubin, G. M. (1990) Isolation and expression of scabrous, a gene regulating neurogenesis in Drosophila. Genes & Development 4, 1848–1861. Nakano, Y.; Guerrero, I.; Hidalgo, A.; Taylor, A.; Whittle, J. R. S.; Ingham, P. W. (1989) A protein with several possible membrane-spanning domains encoded by the Drosophila segment polarity gene patched. Nature 341, 508–513. Noiri, E.; Kuwata, S.; Nosaka, K.; Tokunaga, K.; Juji, T.; Shibata, Y.; Kurokawa, K. (1994) Tumor necrosis factor-! mRNA Expression in lipopolysaccharide-stimulated rat kidney. Am. J. Path. 144 (6), 1159–1166. Ono, J. K.; McCaman, R. E. (1992) In situ hybridization of whole-mounts of Aplysia ganglia using non-radioactive probes. J. Neurosci. Meth. 44, 71–79. Radema, S. A.; van Deventer, S. J. H.; Cerami, A. (1991) Interleukin 1" Is expressed predominantly by enterocytes in experimental colitis. Gastroenterol. 100, 1180–1186. Rao, Yi; Jan, L-Y; Jan, Y. N. (1990) Similarity of the product of the Drosophila neurogenic gene big brain to transmembrane channel proteins. Nature 345, 163–157. Reuter, R.; Leptin, M. (1994) Interacting functions of snail, twist and huckebein during the early development of germ layers in Drosophila. Develop. 120, 1137–1150. Rothe, M.; Pehl, M.; Taubert, H.; Jäckle, H. (1992) Loss of gene function through rapid mitotic cycles in the Drosophila embryo. Nature 359, 156–159. Sampedro, J.; Guerrero, I. (1991) Unrestricted expression of the Drosophila gene patched allows a normal segment polarity. Nature 353, 187–190. Sano, J.; Miki, K.; Ichinose, M.; Kimura, M.; Kurokawa, K.; Aida, T.; Ishizaki, M.; Asano, G.; Masugi, Y.; Wong, R. N. S.; Takahashi, K. (1989) In situ localization of pepsinogens I and II mRNA in human gastric mucosa. Acta Pathol. Japonica. 39 (12), 765–771.

6

Schlinger, B. A.; Amur-Umarjee, S.; Shen, P.; Campagnoni, A. T.; Arnold, A. P. (1994) Neuronal and non-neuronal aromatase in primary cultures of developing zebra finch telencephalon. J. Neurosci. 14 (12), 7541–7552. Shermoen, A. W.; O’Farrell, P. H. (1991) Progression of the cell cycle through mitosis leads to abortion of nascent transcripts. Cell 67, 303–310. Shin, D. M.; Chiao, P. J.; Sacks, P. G.; Shin, H. J.; Hong, W. K.; Hittelman, W. N.; Tainsky, M. A. (1993) Activation of ribosomal protein S2 gene expression in a hamster model of chemically induced oral carcinogenesis. Carcinogenesis 14 (1), 163–166. Sibon, O. C. M.; Cremers, F. F. M.; Boonstra, J.; Humbel, B. M.; Verkleij, A. J. (1993) Localisation of EGF-receptor mRNA in the nucleus of A431 cells by light microscopy. Cell Biol. Intern. 17 (1), 1–11.

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6

Coates, P. J.; Mak, W. P.; Slavin, G.; d’Ardenne, A. J. (1991) Detection of single copies of Epstein-Barr virus in paraffin wax sections by non-radioactive in situ hybridization. J. Clin. Pathol. 44, 487–491. Dirks, R. W.; Van Dorp, A. G. M.; Van Minnen, J.; Fransen, J. A. M; Van Der Ploeg, M.; Raap, A. K. (1992) Electron microscopic detection of RNA sequences by non-radioactive in situ hybridization in the mollusk lymnaea stagnalis. J. Histochem. Cytochem. 40 (11), 1647–1657. Dirks, R. W.; Van de Rijke, F. M.; Fujishita, S.; Van Der Ploeg, M.; Raap, A. K. (1993) Methodologies for specific intron and exon RNA localization in cultured cells by haptenized and fluorochromized probes. J. Cell Sci. 104, 1187–1197. Flemming, K. A.; Evans, M.; Ryley, K. C.; Franklin, D.; Lovell-Badge, R. H.; Morey, A. L. (1992) Optimization of non-isotopic in situ hybridization on formalin-fixed, paraffin-embedded material using digoxigenin-labeled probes and transgenic tissues. J. Pathol. 167, 9–17. Whawell, S. A.; Wang, Y.; Fleming, K. A.; Thompson, E. M.; Thompson, J. N. (1993) Localization of plasminogen activator inhibitor-1 production in inflamed appendix by in situ mRNA hybridization. J. Pathol. 169, 67–71.

c. cDNA synthesis Ultsch, A.; Schuster, C. M.; Laube, B.; Schloss, P.; Schmitt, B.; Betz, H. (1992) Glutamate receptors of Drosophila melanogaster: Cloning of a kainateselective subunit expressed in the central nervous system. Proc. Natl. Acad. Sci. USA 89, 10484–10488.

d. PCR

Xing, Y.; Johnson, C. V.; Dobner, P. R.; Lawrence, J. B. (1993) Higher level organization of individual gene transcription and RNA splicing. Science 259, 1326–1330.

Barka, T.; van der Noen, H. (1993) Expression of the cysteine proteinase inhibitor cystatin C gene in rat heart: Use of digoxigenin-labeled probes generated by polymerase chain reaction directly for in situ and northern blot hybridizations. J. Histochem. Cytochem. 41 (12) 1863–1867.

Yamada, H.; Aida, T.; Taguchi, K.; Asona, G. (1989) Localization of type III procollagen mRNA in areas of liver fibrosis by in situ hybridization. Acta Pathol. Japonica. 39 (11), 719–724.

Barka, T.; van der Noen, H. (1994) Expressions of the genes for cysteine proteinase inhibitors cystatin C and cystatin S in rat submandibular salivary gland. Archs oral Biol. 39 (4), 307–314.

Zhang, P.; Knowles, B. A.; Goldstein, L. S. B.; Hawley, R. S. (1990) A kinesin-like protein required for distributive chromosome segregation in Drosophila. Cell 62, 1053–1062.

Barka, T.; van der Noen, H. (1994) Expression of the cysteine proteinase inhibitor cystatin C mRNA in rat eye. Anatom. Rec. 239, 343–348.

Zhao, T.-J.; Chiu, T. H.; Rosenberg, H. C. (1994) Reduced expression of #-Aminobutyric acid type A/benzodiazepine receptor #2 and !5 subunit mRNAs in brain regions of flurazepam-treated rats. Mol. Pharmacol. 45, 657–663. Zheng, M. H.; Fan, Y.; Wysocki, S.; Wood, D. J.; Papadimitriou, J. M. (1993) Detection of mRNA for carbonic anhydrase II in human oesteoclast-like cells by in situ hybridization. J. Bone Mineral Res. 8 (1), 113–118.

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CONTENTS

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B. RNA probes Adham, I. M.; Burkhardt, E.; Benahmed, M.; Engel, W. (1993) Cloning of a cDNA for a novel insulin-like peptide of the testicular leydig cells. J. Biol. Chem. 268 (35), 26668–26672. Ahn, K. Y.; Madsen, K. M.; Tisher, C. C.; Kone, B. C. (1993) Differential expression and cellular distribution of mRNAs encoding !- and "-isoforms of Na+-K+-ATPase in rat kidney. Am. J. Physiol. 265, 792–801. Bachmann, S.; Le Hir, M.; Eckardt, K.-U. (1993) Co-localization of erythropoietin mRNA and ecto-5’nucleotidase immunoreactivity in peritubular cells of rat renal cortex indicates that fibroblasts produce erythropoietin. J. Histochem. Cytochem. 41, 335–341. Baldino, F.; Robbins, E.; Grega D.; Meyers, S. L.; Springer, J. E.; Lewis, M. E. (1989) Non-radioactive detection of NGF-receptor messenger RNA with digoxigenin-UTP labeled RNA probes. 19th Annual Meeting Soc. Neuroscience, Phoenix, Arizona, USA, Oct. 29–Nov. 3, 1989. Soc. Neurosci. Abst. 15 (1), 864. Barthel, L. K.; Raymond, P. A. (1993) Subcellular localization of !-tubulin and opsin mRNA in the goldfish retina using digoxigenin-labeled cRNA probes detected by alkaline phosphatase and HRP histochemistry. J. Neurosci. Meth. 50, 145–152. Biffo, S.; Verdun di Cantogno, L.; Fasolo, A. (1992) Double labeling with non-isotopic in situ hybridization and BrdU immunohistochemistry: Calmodulin (CaM) mRNA expression in post-mitotic neurons of the olfactory system. J. Histochem. Cytochem. 40 (4), 535–540.

CONTENTS

INDEX

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6

Campbell, A. M.; Wuytack, F.; Fambrough, D. M. (1993) Differential distribution of the alternative forms of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, SERCA2b and SERCA2a, in the avian brain. Brain Res. 605, 67–76. Canas, L. A.; Busscher, M.; Angenent, G. C.; Beltran, J.-P.; van Tunen, A. J. (1994) Nuclear localization of the petunia MADS box protein FBP1. Plant J. 6 (4), 597–604. Caroni, P.; Schneider, C. (1994) Signaling by insulinlike growth factors in paralyzed skeletal muscle: rapid induction of IGF-1 expression in muscle fibers and prevention of interstitial cell proliferation by IGF-BP5 and IGF-BP4. J. Neurosci. 14 (5), 3378–3388.

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6

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C. Oligonucleotide probes a. 3'-Endlabeling Campbell, A. M.; Wuytack, F.; Fambrough, D. M. (1993) Differential distribution of the alternative forms of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, SERCA2b and SERCA2a, in the avian brain. Brain Res. 605, 67–76. Hodges, E.; Howell, W. M.; Tyacke, S. R.; Wong, R.; Cawley, M. I. D. (1994) Detection of T-Cell receptor " chain mRNA in frozen and paraffin-embedded biopsy tissue using digoxigenin-labeled oligonucleotide probes in situ. J. Pathol. 174, 151–158. McNicol, A. M.; Farquharson, M. A.; Walker, E. (1991) Non-Isotopic in situ hybridization with digoxigenin and alkaline phosphatase labeled oligodeoxynucleotide probes. Path. Res. Pract. 187, 556–558. Roncaroli, F.; Dina, R.; Geuna, M.; Reato, G.; Ponti, R.; Palestro, G. (1994) Identification of granulocyte-macrophage colony stimulating factor receptor mRNA by non-isotopic in situ hybridization in bone marrow biopsies. Haemotol. 79, 322–327. Thomas, G. A.; Davies, H. G.; Williams, E. D. (1993) Demonstration of mRNA using digoxigenin labeled oligonucleotide probes for in situ hybridzation in formamide free conditions. J. Clin. Pathol. 46, 171–174.

b. 3'-Tailing Artero, R. D.; Akam, M.; Pérez-Alonso, M. (1992) Oligonucleotide probes detect splicing variants in situ in Drosophila embryos. Nucl. Acids Res. 20 (21), 5687–5690. Amberg, D. C.; Goldstein, A. L.; Cole, C. N. (1992) Isolation and characterization of RAT1: an essential gene of Saccharomyces cerevisiae required for the efficient nucleocytoplasmic trafficking of mRNA. Genes Develop. 6, 1173–1189. Baldino, F.; Lewis, M. E. (1989) Non-radioactive in situ hybridization histochemistry with digoxigenindUTP labeled oligonucleotides. Methods in Neuroscience Vol. 1 (Conn, P. M., ed.) Acadmic Press, New York, 282–292.

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Dickerson, D. S.; Huerter, B. S.; Morris, S. J.; Chronwall, B. M. (1994) POMC mRNA levels in individual melanotropes and GFAP in glial-like cells in rat pituitary. Peptides 15 (2), 247–256. Dirks, R. W.; Van Dorp, A. G. M.; Van Minnen, J.; Fransen, J. A. M.; Van der Ploeg, M.; Raap, A. K. (1992) Electron microscopic detection or RNA sequences by non-radioactive in situ hybridization in the mollusk lymnaea stagnalis. J. Histochem. Cytochem. 40, 1647–1657. Dirks, R. W.; Van Gijlswijk, R. P. M.; Vooljs, M. A.; Smit, A. B.; Bogerd, J.; Van Minnen, J.; Raap, A. K.; Van der Ploeg, M. (1991) 3’End fluorochromized and haptenized oligonucleotides as in situ hybridization probes for multiple, simultaneous RNA detection. Experim. Cell Res. 194, 310–315. Dirks, R. W.; Van der Rijke, F. M.; Fujishita, S.; Van der Ploeg, M.; Raap, A. K. (1993) Methodologies for specific intron and exon RNA localization in cultured cells by haptenized and fluorochromized probes. J. Cell Sci. 104, 1187–1197. Dumas, S.; Horellou, P.; Helin, C.; Mallet, J. (1992) Co-expression of tyrosine hydroxylase messenger RNA1 and 2 in human ventral mesencephalon revealed by digoxigenin- and biotin-labeled oligodeoxyribonucleotides. J. Chem. Neuroanatomy 5, 11–18. Crabb, I. D.; Hughes, S. S.; Hicks, D. G.; Puzas, J. E.; Tsao, G. J. Y.; Rosier, R. N. (1992) Nonradioactive in situ hybridization using digoxigenin-labeled oligonucleotides. Am. J. Pathol. 141 (3), 579–589. Farquharson, M.; Harvie, R.; McNicol, A. M. (1990) Detection of messenger RNA using a digoxigenin end labeled oligonucleotide probe. J. Clin Pathol. 43, 424–428. Farquharson, M.; Harvie, R.; McNicol, A. M. (1990) Detection of pro-opiomelanocortin messenger RNA using adigoxigenin end labeled oligodeoxynucleotide probe. J. Endocrinol. 124 (Supplement). Fevre-Montange, M.; Trembleau, A.; Landry, M.; Calas, A. (1992) Hybridation in situ avec des sondes oligonucléotidiques marquées à la digoxigénine: application à la détection des ARNm de la vasopressine et de l’ocytocine. Techniques, 479–486.

6

Grega, D.; Cavanaugh, T.; Martin, R.; Lewis, M.; Baldino, F. (1989) In situ hybridization histochemistry using a new non-radioactive detection method. ICSU Short Reports, Advances in Gene Technology: Molecular Neurobiology and Neuropharmacology, Proceedings of the 1989 Miami Bio/Technology Winter Symposium, Vol. 9, p. 69, IRL Press, McLean, Virginia. Guellec Le, P.; Dumas, S.; Volle, G. E.; Pidoux, E.; Moukhtar, M. S.; Treilhou-Lahille, F. (1993) An efficient method to detect calcitonin mRNAin normal and neoplastic rat c-cells (Medullary Thyroid Carcinoma) by in situ hybridization using a digoxigenin-labeled synthetic oligodeoxyribonucleotide probe. J. Histochem. Cytochem. 41, 389–395. Hamada, K.; Okawara, Y.; Fryer, J. N.; Tomonaga, A.; Fukuda, H. (1994) Localization of mRNA of procollagen !1 type I in torn supraspinatus tendons. Clin. Orthopaed. Related Res. 304, 18–21.

184

Harper, S. J.; Pringle, J. H.; Gillies, A.; Allen, A. C.; Layward, L.; Feehally, J.; Lauder, I. (1992) Simultaneous in situ hybridization of native mRNA and immunoglobulin detection by conventional immunofluorescence in paraffin way embedded sections. J. Clin. Pathol. 45, 114–119. Harvie, R.; Elvin, P.; McArdle, C.; Morten, J. E. N.; McNicol, A. M. (1991) Detection of mRNA for ribosomal phosphoprotein P2 in normal colon and colonic tumours by in situ hybridization. J. Pathol. 164, 67–73. Hilton, D. A.; Day; C.; Pringle, J. H.; Fletcher, A.; Chambers, S. (1992) Demonstration of the distribution of Coxsackie virus RNAin neonatal mice by non-isotopic in situ hybridization. J. Virol. Meth. 40, 155–162. Hilton, D. A.; Fletcher, A.; Pringle, J. H. (1994) Absence of coxsacki viruses in idiopathic inflammatory muscle disease by in situ hybridization. Neuropathol. Appl. Neurobiol. 20, 238–242. Humpel, C.; Lindqvist, E.; Olson, L. (1993) Detection of nerve growth factor mRNA in rodent salivary glands with digoxigenin- and 33P-labeled oligonucleotides: effects of castration and sympathectomy. J. Histochem. Cytochem. 41 (5), 703–708. Kendall, C. H.; Roberts, P. A.; Pringle, J. H.; Lauder, I. (1991) The expression of parathyroid hormone messenger RNA in normal and abnormal parathyroid tissue. J. Pathol. 165, 111–118. Kennedy, R. E.; Hutchins, J. B. (1992) Choline acetyltransferase expression studied with an oligonucleotide probe. Cell. Mol. Neurobiol. 12 (4), 309–315. Khan, G.; Coates, P .J.; Kangro, H. O.; Slavin, G. (1992) Epstein Barr virus (EBV) encoded small RNAs: Targets for detection by in situ hybridization with oligonucleotide probes. J. Clin. Pathol. 45, 616–620. Khan, G.; Norton, A. J.; Slavin, G. (1993) EpsteinBarr virus in angioimmunoblastic T-cell lymphomas. Histophatol. 22, 145–149. Kirchgessner, A. L.; Liu, M.-T.; Howard, M. J.; Gershon, M. D. (1993) Detection of the 5-HT1A Receptor and 5-HT1A Receptor mRNA in the rat bowel and pancreas: comparison with 5-HT1P Receptors. J. Comparative Neurol. 327, 233–250. Koji, T.; Brenner, R. M. (1993) Localization of estrogen receptor messenger ribonucleic acid in rhesus monkey uterus by nonradioactive in situ hybridization with digoxigenin-labeled oligodeoxynucleotides. Endocrinol. 132, 382–392. Komminoth, P. (1992) Digoxigenin as an alternative probe labeling for in situ hybridization. Diagn. Mol. Pathol. 1 (2), 142–150. Komminoth, P.; Merk, F. B.; Leav, I.; Wolfe, H. J.; Roth, J. (1992) Comparison of 35S- and digoxigeninlabeled RNA and oligonucleotide probes for in situ hybridization. Histochem. 98, 217–228. Krizbai, I.; Deli, M.; Lengyel, I.; Maderspach, K.; Pákáski, M.; Joó, F.; Wolff, J. R. (1993) In situ hybridization with digoxigenin labeled oligonucleotide probes: detection of CAMK-II gene expression in primary cultures of cerebral endothelial cells. Neurobiol. 1 (3), 235–240.

CONTENTS

INDEX

R eferences

7. Apoptosis research using DIG-dUTP and terminal transferase Billig, H.; Furuta, I.; Hsueh, A. J. (1993) Estrogens inhibit and androgens enhance ovarian granulosa cell apoptosis. Endocrinol. 133 (5) 2204–2212. Billig, H.; Furuta, I.; Hsueh, A. J. W. (1994) Gonadotropin-releasing hormone directly induces apoptotic cell death in the rat ovary: Biochemical and in situ detection of deoxyribonucleic acid fragmentation in granulosa cells. Endocrinol. 134 (1), 245–252. Gold; R.; Schmied, M.; Rothe, G.; Zischler, H.; Breitschopf, H.; Wekerle, H.; Lassmann, H. (1993) Detection of DNA fragmentation in apoptosis: Application of in situ nick translation to cell culture systems and tissue sections. J. Histochem. Cytochem. (in press).

Gorczyca, W.; Gong, J.; Ardelt, B.; Traganos, F.; Darzynkiewicz, Z. (1993) The cell cycle related differences in susceptibility of HL-60 cells to apoptosis induced by various antitumor agents. Cancer Res. 53, 3186–3192. Gorczyca, W.; Gong, J.; Darzynkiewicz, Z. (1993) Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Canc. Res. 53, 1945–1951. Sgonc, R.; Boeck, G.; Dietrich, H.; Gruber, J.; Reicheis, H.; Wick, G. (1994) Simultaneous determination of cell surface antigens and apoptosis. TIG 10 (2), 41–42.

Gorczyca, W.; Bigman, K.; Mittelman, A.; Ahmed, T.; Gong, J.; Melamed, M. R.; Darzynkiewicz, Z. (1993) Induction of DNA strand breaks associated with apoptosis during treatment of leukemias. Leukemia 7 (5), 659–670.

8. Miscellaneous Schöfer, C.; Müller, M.; Leitner, M. D.; Wachtler, F. (1993) The uptake of uridine in the nucleolus occurs in the dense fibrillar component. Cytogenet. Cell Genet. 64, 27–30. Wansink, D. G.; Manders, E. E. M.; van der Kraan, I.; Aten, J. A.; van Driel, R.; de Jong, L. (1994) RNA polymerase II transcription is concentrated outside replication domains throughout S-phase. J. Cell Sci. 107, 1449–1456.

6

200

CONTENTS

INDEX

R eferences

Krusche, C.; Beier, H. M. (1994) Localization of uteroglobin mRNA by nonradioactive in situ hybridization in the pregnant rabbit endometrium. Ann. Anat. 176, 23–31. Laverdure, A.-M.; Carette-Desmoucelles, C.; Breuzet, M.; Descamps, M. (1994) Neuropeptides and related nucleic acid sequences detected in peneid shrimps by immunohistochemistry and molecular hybridizations. Neurosci. 60 (2), 569–579. Laverdure, A.-M.; Breuzet, M.; Soyez, D.; Becker, J. (1992) Detection of the mRNA encoding vitellogenesis inhibiting hormone in neurosecretory cells of the X-organ in homarus americanus by in situ hybridization. Gen. Compar. Endocrinol. 87, 443–450. Lee, D.; Huang, W.; Yang, Z.; Copolov, D. L.; Lim, A. T. (1992) In vitro evidence for modulation of morphological and functional development of hypothalamic immunoreactive atrial natriuretic peptide neurons by cyclic 3', 5'-adenosine monophosphate. Endocrinol. 131, 911–918.

Myatt, N.; Coghill, G.; Morrison, K.; Jones, D.; Cree, I. A. (1994) Detection of tumour necrosis factor-! in sarcoidosis and tuberculosis granulomas using in situ hybridization. J. Clin. Pathol. 47, 423–426. Nakagawa, M.; Kukita, T.; Nakasima, A.; Kurisu, K. (1994) Expression of the type I collagen gene in rat periodontal ligament during tooth movement as revealed by in situ hybridization. Archs oral Biol. 39 (4), 289–294. Negro, F.; Papotti, M.; Pacchioni, D.; Galimi, F.; Bonino, F.; Bussolati, G. (1994) Detection of human androgen receptor mRNA in hepatocellular carcinoma by in situ hybridization. Liver 14, 213–219. Nicoloso, M.; Caizergues-Ferrer, M.; Michot, B.; Azum, M.-C.; Bachellerie, J.-P. (1994) U20, a novel small nucleolar RNA, is encoded in an intron of the nucleolin gene in mammals. Mol. Cell. Biol. 14 (9), 5766–5776.

Lewis, M. E.; Robbins, E.; Grega, D.; Baldino, F. (1989) Nonradioactive detection of vasopression and somatostatin mRNA with Digoxigenin-labeled oligonucleotide probes. Ann. NY Acad. Sci. V: Molecular Biology: The Future of Neuropeptide Research, pp 246–253.

O’Keefe, R. J.; Puzas, J. E.; Loveys, L.; Hicks, D. G.; Rosier, R. N. (1994) Analysis of type II and type X collagen synthesis in cultured growth plate chondrocytes by in situ hybridization: rapid induction of type X collagen in culture. J. Bone Mineral Res. 9 (11), 1713–1722.

Matute, C.; Wahle, P.; Gutiérrez-Igarza, K.; Albus, K. (1993) Distribution of neurons expression substance P receptor messenger RNA in immature and adult cat visual cortex. Exp. Brain Res. 97, 295–300.

Ong, W. Y.; Garey, L. J.; Sumi, Y. (1994) Distribution of preprosomatostatin mRNA in the rat parietal and temporal cortex. Mol. Brain Res. 23, 151–156.

Mertani, H. C.; Waters, M. J.; Jambou, R.; Gossard, F.; Morel, G. (1994) Growth hormone receptor inding protein in rat anterior pituitary. Neuroendocrinol. 59, 483–494. Miranda, R. C.; Toran-Allerand, C. D. (1992) Developmental expression of estrogen receptor mRNA in the rat cerebral cortex: A nonisotopic in situ hybridization histochemistry study. Cerebral Cortex Jan/Feb 2, 1–15. Mitchel, V.; Gambiez, A.; Beauvillain, J. C. (1993) Fine-structural localization of proenkephalin mRNAs in the hypothalamic magnocellular dorsal nucleus of the guinea pig: a comparison of radioisotopic and enzymatic in situ hybridization methods at the lightand electron-microscopic levels. Cell Tissue Res. 274, 219–228. Millar, M. R.; Sharpe, R. M.; Maguire, S. M.; Saunders, P. T. K. (1993) Cellular localisation of messenger RNAs in rat testis: application of digoxigenin-labeled ribonucleotide probes to embedded tissue. Cell Tissue Res. 273, 269–277. Miyazaki, M.; Nikolic-Paterson, D. J.; Masayuki, E.; Nomoto, Y.; Sakai, H.; Atkins, R. C.; Koji, T. (1994) A sensitive method of non-radioactive in situ hybridization for mRNA localization within human renal biopsy specimens: use of digoxigenin labeled oligonucleotides. Int. Med. 33, 87–91. Murray, G. I.; Paterson, P. J.; Ewen, S. W. B.; Melvin, W. T. (1992) In situ hybridization of albumin mRNA in normal liver and hepatocellular carcinoma with a digoxigenin labeled oligonucleotide probe. J. Clin. Pathol. 45, 21–24.

CONTENTS

INDEX

Pacchioni, D.; Papotti, M.; Andorno, E.; Bonino, F.; Mondardini, A.; Oliveri, F.; Brunetto, M.; Ussolati, G.; Negro, F. (1993) Expression of estrogen receptor mRNA in tumorous and non-tumorous liver tissue as detected by in situ hybridization. J. Surgical Oncol. Suppl. 3, 14–17. Papotti, M.; Pacchioni, D.; Negro, F.; Bonino, F.; Bussolati, G. (1994) Albumin gene expression in liver tumors: diagnostic interest in fine needle aspiration biopsies. Modern Pathol. 7 (3), 271–275. Paulmyer-Lacroix, O.; Anglade, G.; Grino, M. (1994) Insulin-induced hypoglycaemia increase colocalization of corticotrophin-releasing factor and arginine vasopressin mRNAs in the rat hypothalamic paraventricular nucleus. J. Mol. Endocrinol. 13, 313–320.

6

Samoszuk, M.; Nansen, L. (1990) Detection of Interleukin-5 messenger RNA in Reed-Sternberg cells of Hodgkin’s desease with eosinophilia. Blood 75 (1), 13–16. Sharara, F. I.; Nieman, L. K. (1994) Identification and cellular localization of growth hormone receptor gene expression in the human ovary. J. Clin. Endocrinol. Metabolism 79 (2), 670–672. Shorrock, K.; Roberts, P.; Pringle, J. H.; Lauder, I. (1991) Demonstration of insulin and glucagon mRNA in routinely fixed and processed pancreatic tissue by in situ hybridization.J. Pathol. 165, 105–110. Thomas, G. A.; Davies, H. G.; Williams, E. D. (1994) Site of production of IGF1 in the normal and stimulated mouse thyroid. J. Pathol. 173, 355–360.

185

R eferences

Throsby, M.; Lee, D.; Huang, W.; Yang, Z.; Copolov, D. L.; Lim, A. T. (1991) Evidence for atrial natriuretic peptide-(5-28) production by macrophages of the rat spleen: An immunochemical and nonradioactive in situ hybridization approach. Endo. 129, 991–1000. Throsby, M.; Yang, Z.; Lee, D.; Huang, W.; Copolov, D. L.; Lim, A. T. (1993) In vitro evidence for atrial natriuretic factor-(5-28) production by macrophages of adult rat thymi. Endocrinol. 132 (5), 2184–2190. Tokushige, E.; Ito, K.; Ushikai, M.; Katahira, S.; Fukuda, K. (1994) Localization of IL-1" mRNA and cell adhesion molecules in the maxillary sinus mucosa of patients with chronic sinusitis. Laryngoscope 104, 1245–1250. Toran-Allerand, C. D.; Miranda, R. C.; Hochberg, R. B.; MacLusky, N. J. (1992) Cellular variations in estrogen receptor mRNA translation in the developing brain: evidence from combined [125I] estrogen autoradiography and non-isotopic in situ hybridization histochemistry. Brain Res. 576, 25–41. Trembleau, A.; Roche, D.; Calas, A. (1993) Combination of non-radioactive and radioactive in situ hybridization with immunohistochemistry: a new method allowing the simultaneous detection of two mRNAs and one antigen in the same brain tissue section. J. Histochem. Cytochem. 41, 489–498. Trembleau, A.; Morales, M.; Bloom, F. E. (1994) Aggregation of vasopressin mRNA in a subset of axonal swellings of the median eminence and posterior pituitary: Light and electron microscopic evidence. J. Neurosci. 14 (1), 39–53. Ueyama, T.; Houtani, T.; Nakagawa, H.; Baba, K.; Ikeda, M.; Yamashita, T.; Sugimoto, T. (1994) A subpopulation of olivocerebellar projection neurons express neuropeptide Y. Brain Res. 634, 353–357. Wani, G.; Wani, A. A.; Ambrosio, S. (1992) In situ hybridization of human kidney tissue reveals celltype-specific expression of the O6-methylguanineDNA methyltransferase gene. Carcinogenesis 13 (3), 463–468. Wani, G.; Wani, A. A.; Ambrosio, S. (1993) Cell typespecific expression of the O6-alkylguanine-DNA alkyltransferase gene in normal human liver tissues as revealed by in situ hybridization. Carcinogenesis 14 (4), 737–741.

6

Whitman, G. F.; Pantazis, C. G. (1991) Cellular localization of müllerian inhibiting substance messenger ribonucleic acid during human ovarian follicular development. Am. J. Obstet. Gynecol. 165, 1881–1886. Woodroofe, M. N.; Cuzner, M. L. (1993) Cytokine mRNA expression in inflammatory multiple sclerosis lesions: detection by non-radioactive in situ hybridization. Cytokine 5 (6), 583–588.

Yamada, S.; Takahashi, M.; Hara, M.; Sano, T.; Aiba, T.; Shishiba, Y.; Suzuki, T.; Asa, S. L. (1994) Growth hormone and prolactin gene expression in human densely and sparsely granulated somatotroph adenomas by in situ hybridization with digoxigeninlabeled probes. Diagn. Mol. Pathol. 3 (1), 46–52. Young, W. S. III (1989) Simultaneous use of digoxigenin- and radiolabeled oligodeoxyribonucleotide probes for hybridization histochemistry. Neuropeptides (Edinburgh) 18, 271–275. Young, W. S. III; Horvath, S.; Palkovits, M. (1990) The influence of hyperosmolality and synaptic inputs on galanin and vasopressin expression in the hypothalamus. Neurosci. 39 (1), 115–125. Young, W. S. III; Reynolds, K.; Shepard, E. A.; Gainer, H.; Castel, M. (1990) Cell-specific expression of the rat oxytocin gene in transgenic mice. J. Neuroendocrinol. 2 (6), 917–925. Zupanc, G. K. H.; Okawara, Y.; Zupanc, M. M.; Fryer, J. N.; Maler, L. (1991) In situ hybridization of putative somatostatin mRNA in the brain of electric gymnotiform fish. Neuro Report 2, 707–710.

c. Chemical synthesis Hassall, C. J. S.; Stanford, S. C.; Burnstock, G.; Buckley, N. J. (1993) Co-expression of four muscarinic receptor genes by the intrinsic neurons of the rat and guinea-pig heart. Neurosci. 56 (4), 1041–1048. Hell, K.; Pringle, J. H.; Hansmann, M.-L.; Lorenzen, J.; Colloby, P.; Lauder, I.; Fischer, R. (1993) Demonstration of light chain mRNA in hodgkin’s disease.J. Pathol. 171, 137–143. Hodges, E.; Howell, W. M.; Tyacke, S. R.; Wong, R.; Cawley, M. I. D.; Smith, J. L. (1994) Detection of T-cell receptor " chain mRNAin frozen and paraffinembedded biopsy tissue using digoxigenin-labeled oligonucleotide probes in situ. J. Pathol. 174, 151–158. Li, D.; Bell, J.; Brown, A.; Berry, C. L. (1994) The observation of angiogenin and basic fibroblast growth factor gene expression in human colonic adenocarcinomas, gastric adenocarcinomas, and hepatocellular carcinomas. J. Pathol. 172, 171–175. Shanks, J. H.; Lappin, T. R. J.; Hill, C. M. In situ hybridization for erythropoietin messenger RNA using digoxigenin-labeled oligonucleotides. Annals New York Academy of Sciences. Thomas, G. A.; Neonakis, E.; Davies, H. G.; Wheeler, M. H.; Williams, E. D. (1994) Synthesis and storage in rat thyroid C-cells. J. Histochem. Cytochem. 42 (8), 1055–1060.

Xerri, L.; Monges, G.; Guigou, V.; Parc, P.; Hassoun, J. (1992) Detection of gastrin mRNA by in situ hybridization using radioactive- and digoxigeninlabeled probes: a comparative study. APMIS 100, 949–953.

186

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1.2 rRNA target

1.3 RNA target

A. RNA probe

A. Oligonucleotide Probes

Cary, S. C.; Warren, W.; Anderson, E.; Giovannoni, S. J. (1993) Identification and localization of bacterial endosymbionts in hydrothermal vent taxa with symbiont-specific polymerase chain reaction amplification and in situ hybridization techniques. Mol. Marine Biol. Biotechn. 2 (1), 51–62.

b. 3'-Tailing

Fischer, D.; Weisenberger, D.; Scheer, U.: Assigning functions to nucleolar structures. Chomosoma Focus.

B. Oligonucleotide probes

Negro, F.; Pacchioni, D.; Shimizu, Y.; Miller, R. H.; Bussolati, G.; Purcell, R. H.; Bonino, F. (1992) Detection of intrahepatic replication of hepatitis C virus RNA by in situ hybridization and comparison with histopathology. Proc. Natl. Acad. Sci. USA 89, 2247–2251. Azum-Gelade, M.-C.; Noaillac-Depeyre, J.; Caizergues-Ferrer, M.; Gas, N. (1994) Cell cycle redistribution of U3 snRNA and fibrillarin. J. Cell Sci. 107, 463–475.

a. 3'-Endlabeling Dixon, B. (1992) Targeting ribosomal RNA: Identifying microbes. BioTechn. 10, 124. Zarda, B.; Amann, R.; Wallner, G.; Schleifer, K.-H. (1991) Identification of single bacterial cells using digoxigenin-labeled rRNA-targeted oligonucleotides. J. Gen. Microbiol. 137, 2823–2830.

b. 3'-Tailing

B. RNA probes Bashir, R.; McManus, B.; Cunningham, C.; Weisenburger, D.; Hochberg, F. (1994) Detection of Eber-1 RNA in primary brain lymphomas in immunocompetent and immunocompromised patients. J. Neuro-Oncol. 20, 47–53. Di Giuseppe, J. A.; Wu, T.-C.; Corio, R. L. (1994) Analysis of epstein-barr virus-encoded small RNA 1 expression in benign lymphoepithelial salivary gland lesions. Mod. Pathol. 7 (5), 555–559.

Azum-Gélade, M.-C.; Noaillac-Depeyre, J.; Caizergues-Ferrer, M.; Gas, N. (1994) Cell cycle redistribution of U3 snRNA and fibrillarin. Presence in the cytoplasmic nucleolus remnant and in the prenucleolar bodies at telophase. J. Cell Sci. 107, 463–475.

Lima, M. I.; Fonseca, M. E. N.; Flores, R.; Kitajima, E. W. (1994) Detection of avocado sunblotch viroid in chloroplasts of avocado leaves by in situ hybridization. Arch. Virol. 138, 385–390.

Concha, I. I.; Urzua, U.; Yanez, A.; Schroeder, R.; Pessot, C.; Burzio, L. O. (1993) U1 and U2 snRNA are localized in the sperm nucleus. Experiment. Cell Res. 204, 378–381.

Lin, N.-S.; Chen, C.-C.; Hsu, Y.-H. (1993) Postembedding in situ hybridization for localization of viral nucleic acid in ultra-thin sections. J. Histochem. Cytochem. 41 (10), 1513–1519.

c. Chemical synthesis Gersdorf, H.; Pelz, K.; Göbel, U. B. (1993) Fluorescence in situ hybridization for direct visualization of Gram-negative anaerobes in subgingival plaque samples. FEMS Immunol. & Medical Microbiol. 6, 109–114. Roller, C.; Wagner, M.; Amann, R.; Ludwig, W.; Schleifer, K.-H. (1994) In situ probing of Grampositive bacteria with high DNA G+C content using 23S rRNA-targeted oligonucleotides. Microbiol. 140, 2849–2858.

C. DNA probes

6

d. PCR Tanaka, Y.; Enomoto, N.; Kojima, S.; Tang, L.; Goto, M.; Marumo, F.; Sato, C. (1993) Detection of hepatitis C virus RNA in the liver by in situ hybridization. Liver 13, 203–208.

Wallner, G.; Amann, R.; Beisker, W. (1993) Optimizing fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms. Cytom. 14, 136–143. Zarda, B.; Amann, R.; Wallner, G.; Schleifer, K.-H. (1991) Identification of single bacterial cells using digoxigenin-labeled rRNA-targeted oligonucleotides. J. Gen. Microbiol. 137, 2823–2830.

CONTENTS

INDEX

187

R eferences

2. DNA target A. DNA probes a. RPL Boland, N. I.; Gosden, R. G. (1994) Clonal analysis of chimaeric mouse ovaries using DNA in situ hybridization. J. Reprod. Fertility 100, 203–210. Brown, D. W.; Welsh, R. M.; Like, A. A. (1993) Infection of peripancreatic lymph nodes but not islets precedes kilham rat virus-induced diabetes in BB/Wor rats. J. Virol. 67 (10), 5873–5878. Burnett, S.; Kiessling, U.; Pettersson, U. (1989) Loss of bovine papillomavirus DNA replication control in growth-arrested transformed cells. J. Virol. 63 (5), 2215–2225. Cenacchi, G.; Musiani, M.; Gentilomi, G.; Righi, S.; Zerbini, M.; Chandler, J. G.; Scala, C.; La Plaga, M.; Martinelli, G. N. (1993) In situ hybridization at the ultrastructural level: localization of cytomegalovirus DNA using digoxigenin labeled probes. J. Submicrosc. Cytol. Pathol. 25 (3), 341–345. Clark, J.; Moore, L.; Krasinskas, A.; Way, J.; Battey, J.; Tamkun, J.; Kahn, R. A. (1993) Selective amplification of additional members of the ADPribosylation factor (ARF) family: Cloning of additional human and drosophila ARF-like genes. Proc. Natl. Acad. Sci. USA 90, 8952–8956. Coates, P. J.; D’Ardenne, A. J.; Slavin, G.; Kingston, J. E.; Malpas, J. S. (1993) Detection of Epstein-Barr virus in reed-sternberg cells of hodgkin’s disease arising in children. Med. Pediatric Oncol. 21, 19–23. Delvenne, P.; Fontaine, M.-A.; Delvenne, C.; Nikkels, A.; Boniver, J. (1994) Detection of human papillomaviruses in paraffin-embedded biopsies of cervical intraepithelial lesions: analysis by immunohistochemistry, in situ hybridization, and the polymerase chain reaction. Modern Pathol. 7 (1), 113–119. Delvenne, P.; Kaschten, B.; Deneufbourg, J. M.; Demanez, L.; Stevenaert, A.; Reznik, M.; Boniver, J. (1993) Detection of Epstein-Barr virus in a case of undifferentiated nasopharyngeal carcinoma by in situ hybridization with digoxigenin-labeled PCRgenerated probes. Virchows Archiv A. Pathol. Anat. 423, 145–150.

6

Fletcher, S.; Darragh, D.; Fan, Y.; Grounds, M. D.; Fischer, C. J.; Beilharz, M. W. (1993) Specific cloning of DNA fragments unique to the dog Y chromosome. GATA 10 (3–4), 77–83. Furuta, Y.; Inuyama, Y.; Nagashima, K. (1989) Detection of human papillomavirus genome in nasolaryngeal papillomas using digoxigenin labeled DNA probes. J. Otolaryngol. Japan. 92, 2055–2063. Furuta, Y.; Shinohara, T.; Sano, K.; Meguro, M.; Nagashima, K. (1990) In situ hybridization with digoxigenin-labeled DNA probes for detection of viral genomes. J. Clin. Pathol. 43, 806–809.

188

Gentilomi, G.; Musiani, M.; Zerbini, M.; Gallinella, G.; Gibellini, D.; La Placa, M. (1989) A hybrido-immunocytochemical assay for the in situ detection of cytomegalovirus DNA using digoxigenin-labeled probes. J. Immunol. Methods 125, 177–183. Genitilomi, G.; Musiani, M.; Zerbini, M.; Gibellini, D.; Gallinella, G.; Venturoli, S. (1992) Double in situ hybridization for detection of herpes simplex virus and cytomegalovirus DNA using non-radioactive probes. J. Histochem. Cytochem. 40 (3), 421–425. Gentilomi, G.; Zerbini, M.; Musiani, M.; Gallinella, G.; Gibellini, D.; Venturoli, S.; Re, M. C.; Pileri, S.; Finelli, C.; La Placa, M. (1993) In situ detection of B19 DNA in bone marrow of immunodeficient patients using a digoxigenin-labeled probe. Mol. Cell. Probes 7, 19–24. Han, K.-H.; Hollinger, F. B.; Noonan, C. A.; Yoffe, B. (1992) Simultaneous detection of HBV-specific antigens and DNA in paraffin-embedded liver tissue by immunohisto-chemistry and in situ hybridization using a digoxigenin-labeled probe. J. Virol. Meth. 37, 89–98. Heiles, H. B. J.; Genersch, E.; Kessler, C.; Neumann, R.; Eggers, H. J. (1988) In situ hybridization with digoxigenin-labeled DNA of human papillomaviruses (HPV 16/18) in HeLa and SiHa cells. BioTechn. 6, 978–981. Heino, P.; Hukkanen, V.; Arstila, P. (1989) Detection of human papilloma virus (HPV) DNA in genital biopsy specimens by in situ hybridization with digoxigenin-labeled probes. J. Virol. Methods 26, 331–338. Holm, R.; Karlsen, F.; Nesland, J. M. (1992) In situ hybridization with nonisotopic probes using different detection systems. Modern Pathol. 5 (3), 315–319. Han, K. H.; Hollinger, F. B.; Noonan, C. A.; Solomon, H.; Klintmalm, G. B. G.; Genta, R. M.; Yoffe, B. (1993) Southern-blot analysis and simultaneous in situ detection of hepatitis B virus-associated DNA and antigens in patients with end-stage liver disease. Hepatol. 18, 1032–1038. Jackwood, D. J.; Swayne, D. E.; Fisk, R. J. (1992) Detection of infectious bursal disease viruses using in situ hybridization and non-radioactive probes. Avian Diseases 36, 154–157. Jacob, J.; Kassir, R.; Kelsoe, G. (1991) In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. I. The architecture and dynamics of responding cell populations. J. Exp. Med. 173, 1165–1175.

CONTENTS

INDEX

R eferences

Kawase, M.; Honda, M.; Niimura, M. (1994) Detection of human papillomavirus type 60 in plantar cysts and verruca plantaris by the in situ hybridization method using digoxigenin labeled probes. J. Dermatol. 21, 709–715. Keighren, M.; West, J. D. (1993) Analysis of cell ploidy in histological sections of mouse tissues by DNA-DNA in situ hybridization with digoxigeninlabeled probes. Histochem. J. 25, 30–44. Konkle, B. A.; Shapiro, S. S.; Asch, A. S.; Nachman, R. L. (1990) Cytokine-enhanced expression of glycoprotein Iba in human endothelium. J. Biol. Chem. 265 (32), 19833–19838. Lassus, J.; Niemi, K.-M.; Marjamäki, A.; Syrjänen, S.; Kartamaa, M.; Lehmus, A.; Krohn, K.; Ranki, A. (1992) Comparison of four in situ hybridization methods, based on digoxigenin- and biotin-labeled probes, in detecting HPV DNA in male condylomata acuminata. Intern. J. STD & AIDS 3, 196–203. Lau, J. Y. N.; Naoumov, N. V.; Alexander, G. J. M.; Williams, R. (1991) Rapid detection of hepatitis B virus DNA in liver tissue by in situ hybridization and its combination with immunohistochemistry for simultaneous detection of HBV antigens. J. Clin. Pathol. 44, 905–908. Lereuz, M.; Pallier, C.; Vassias, I.; Elouet, J. F.; Romeo, P.; Morinet, F. (1994) Differential transcription, without replication, of non-structural and structural genes of human parvovirus B19 in the UT7/EPO cell line as demonstrated by in situ hybridization. J. Gen. Virol. 75, 1475–1478. Morey, A. L.; Ferguson, D. J. P.; Leslie, K. O.; Taatjes, D. J.; Fleming, K. A. (1993) Intracellular localization of parvovirus B19 nucleic acid at the ultrastructural level by in situ hybridization with digoxigenin-labeled probes. Histochem. J. 25, 421–429. Morris, R. G.; Arends, M. J.; Bishop, P. E.; Sizer, K.; Duvall, E.; Bird, C. C. (1990) Sensitivity of digoxigenin- and biotin-labeled probes of detection of human papillomavirus by in situ hybridization. J. Clin. Pathol. 43, 800–805. Musiani, M.; Gentilomi, G.; Zerbini, M.; Gibellini, D.; Gallinella, G.; Pileri, S.; Baglioni, P.; La Placa, M. (1990) In situ detection of cytomegalovirus DNA in biopsies of AIDS patients using a hybridoimmunocytochemical assay. Histochem. 94, 21–25. Prelle, A.; Fagiolari, G.; Checcarelli, N.; Moggio, M.; Battistel, A.; Comi, G. P.; Bazzi, P.; Bordoni, A.; Zeviani, M.; Scarlato, G. (1994) Mitochondrial myopathy: correlation between oxidative defect and mitochondrial DNA deletions at single fiber level. Acta Neuropathol. 87, 371–376. Permeen, A. M. Y.; Sam, C. K.; Pathmanathan, R.; Prasad, U.; Wolf, H. (1990) Detection of Epstein Barr Virus DNA in nasopharyngeal carcinoma using a non-radioactive digoxigenin-labeled probe. J. Virol. Meth. 27 (3), 261–268.

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INDEX

Premoli-de-Percoco, G.; Galindo, I.; Ramirez, J. L. (1992) In situ hybridization with digoxigenin-labeled DNA probes for the detection of human papillomavirus-induced focal epithelial hyperplasia among Venezuelans. Virchows Archiv A. Pathol. Anat. 420, 295–300. Schmidt, P.; Meyer, H.; Hübert, P.; Hafner, A.; Andiel, E.; Grabner, A.; Dahme, E. (1994) In situ hybridization for demonstration of equine herpesvirus type 1 DNA in paraffin wax-embedded tissues and its use in horses with disseminated necrotizing myeloencephalitis. J. Comp. Path. 110, 215–225. Schwarz, T. F.; Nerlich, A.; Hottenträger, B.; Jäger, G.; Wiest, I.; Kantimm, S.; Roggendorf, H.; Schultz, M.; Gloning, K.-P.; Schramm, T.; Holzgreve, W.; Roggendorf, M. (1991) Parvovirus B19 infection of the fetus. Histology and in situ hybridization. Am. J. Clin. Pathol. 96 (1), 121–126. Schwarz, T. F.; Nerlich, A.; Hillemanns, P. (1993) Detection of parvovirus B19 in fetal autopsies. Arch. Gynecol. Obstet. 253, 207–213. Smith, K. L.; Robbins, P. D.; Dawkins, H. J. S.; Papadimitriou, J. M.; Redmond, S. L.; Carrello, S.; Harvey, J. M.; Sterrett, G. F. (1994) c-erbB-2 amplification in breast cancer: Detection in formalin-fixed, paraffin-embedded tissue by in situ hybridization. Hum. Pathol. 25, 413–418. Thompson, C. H.; Biggs, I. M.; De Zwart-Steffe, R. T. (1990) Detection of molluscum contagiosum virus DNA by in situ hybridization. Pathol. 22, 181–186.

b. Nick translation Coates, P. J.; Mak, W. P.; Slavin, G.; D’Ardenne, A. J. (1991) Detection of single copies of Epstein-Barr virus in paraffin wax sections by non-radioactive in situ hybridization. J. Clin. Pathol. 44, 487–491. Coates, P. J.; Slavin, G.; D’Ardenne, A. J. (1991) Persistence of epstein-barr virus in reed-sternberg cells throughout the course of hodgkin’s disease. J. Pathol. 164, 291–297. Cooper, K.; Herrington, C. S.; Graham, A. K.; Evans, M. F.; McGee, J. O’D (1991) In situ human papillomavirus (HPV) genotyping of cervical intraepithelial neoplasia in South African and British patients: Evidence for putative HPV integration in vivo. J. Clin. Pathol. 44, 400–405.

6

Cooper, K.; Herrington, C. S.; Lo, E. S.-F.; Evans, M. F.; McGee, J. O. (1992) Integration of human papillomavirus types 16 and 18 in cervical adenocarcinoma. J. Clin. Pathol. 45, 382–384. Del Buono, R.; Fleming, K. A.; Morey, A. L.; Hall, P. A.; Wright, N. A. (1992) A nude mouse xenograft model of fetal intestine development and differentiation. Development 114, 67–73. Egawa, K.; Shibasaki, Y.; De Villiers, E.-M. (1993) Double infection with human papillomavirus 1 and human papillomavirus 63 in single cells of a lesion displaying only an human papillomavirus 63-induced cytopathogenic effect. Lab. Invest. 69 (5), 583–588.

189

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Hegele, R. G.; Bicknell, S. G.; Bailey, D. J.; Cameron, R. G. (1994) In situ hybridization for the Y chromosome reveals a donor origin for a posttransplant lymphoproliferative disorder in a sex-mismatched hepatic allograft. Arch. Pathol. Lab. Med. 118, 795–796.

Lawrence, J. B.; Marselle, L. M.; Byron, K. S.; Johnson, C. V.; Sullivan, J. L.; Singer, R. H. (1990) Subcellular localization of low-abundance human immunodeficiency virus nucleic acid sequences visualized by fluorescence in situ hybridization. Proc. Natl. Acad. Sci. 87, 5420–5424.

Heiskanen, M.; Karhu, R.; Hellsten, E.; Peltonen, L.; Kallioniemi, O. P.; Palotie, A. (1994) High resolution mapping using fluorescence in situ hybridization to extended DNA fibers prepared from agaroseembedded cells. BioTechn. 17 (5), 928–933.

Leitch, I. J.; Leitch, A. R.; Heslop-Harrison, J. S. (1991) Physical mapping of plant DNA sequences by simultaneous in situ hybridization of two differently labeled fluorescent probes. Genome 34, 329–333.

Herrington, C. S.; Burns, J.; Graham, A. K.; Bhatt, B.; McGee, J. O. (1989) Interphase cytogenetics using biotin and digoxigenin labeled probes. II. Simultaneous differential detection of human and papilloma virus nucleic acids in individual nuclei. J. Clin. Pathol. (Lond) 42 (6), 601–606.

Morey, A. L.; Porter, H. J.; Keeling, J. W.; Fleming, K. A. (1992) Non-isotopic in situ hybridization and immunophenotyping of infected cells in the investigation of human fetal parvovirus infection. J. Clin. Pathol. 45, 673–678.

Herrington, C. S.; Burns, J.; Graham, A. K.; Evans, M.; McGee, J. O. (1989) Interphase cytogenetics using biotin and digoxigenin labeled probes. I. Relative sensitivity of both reporter molecules for detection of HPV16 in caski cells. J. Clin. Pathol. (Lond) 42 (6), 592–600. Herrington, C. S.; Bhatt, B.; Burns, J.; Graham, A. K.; McGee, J. O. (1989) Simultaneous non-isotopic in situ hybridization using biotinylated and digoxigeninlabeled probes. J. Pathol. (Edin) 157, 163. Herrington, C. S.; De Angelis, M.; Evans, M. F.; Troncone, G.; McGee, J. O. (1992) Detection of high risk human papillomavirus in routine cervical smears: Strategy for screening. J. Clin. Pathol. 45, 385–390. Herrington, C. S.; Graham, A. K.; Burns, J.; McGee, J. O. (1989) Enhancement of sensitivity of digoxigenin labeled probes: the evaluation of a biotin independent detection system. J. Pathol. (Edin) 158, 337. Herrington, C. S.; Graham, A. K; McGee, J. O. (1991) Interphase cytogenetics using biotin and digoxigenin labeled probes: III. Increased sensitivity and flexibility for detecting HPV in cervical biopsy specimens and cell lines. J. Clin. Pathol. 44 (1), 33–38.

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Kerstens, H. M. J.; Poddighe, P. J.; Hanselaar, A. G. J. M. (1995) A novel in situ hybridization signal amplification method based on the deposition of biotinylated tyramine. J. Histochem. Cytochem. 43 (4), 347–352. Kerstens, H. M. J.; Poddighe, P. J.; Hanselaar, A. G. J. M. (1994) Double-target in situ hybridization in brightfield microscopy. J. Histochem. Cytochem. 42 (8), 1071–1077.

190

Scherthan, H. (1991) Characterisation of a tandem repetitive sequence cloned from the deer capreolus capreolus and it’s chromosomal localisation in two muntjac species. Hereditas (in press). Steilen, H.; Ketter, R.; Romanakis, K.; Zwergel, T.; Unteregger, G.; Bonkhoff, H.; Seitz, G.; Ziegler, M.; Zand, K. D.; Wullich, B. (1994) DNA aneuploidy in prostatic adenocarcinoma: A frequent event as shown by fluroescence in situ DNA hybridization. Hum. Pathol. 25, 1306–1313. Troncone, G.; Herrington, C. S.; Cooper, K.; De Angelis, M. L.; McGee, J. O. (1992) Detection of human papillomavirus in matched cervical smears and biopsy specimens by non-isotopic in situ hybridization. J. Clin. Pathol. 45, 308–313. Wachtler, F.; Schöfer, C.; Schedle, A.; Schwarzacher, H. G.; Hartung, M.; Stahl, A.; Gonzales, I.; Sylvester, J. (1991) Transcribed and nontranscribed parts of the human ribosomal gene repeat show a similar pattern of distribution in nucleoli. Cytogenet. Cell Genet. 57, 175–178. Wood, N. B.; Sheikholeslami, M.; Pool, M.; Coon, J. S. (1994) PCR production of a digoxigenin-labeled probe for the detection of human cytomegalovirus in tissue sections. Diagn. Mol. Pathol. 3 (3), 200–208.

d. PCR Saeki, K.; Mishima, K.; Horiuchi, K.; Hirota, S.; Nomura, S.; Kitamura, Y.; Aozasa, K. (1993) Detection of low copy numbers of epstein-barr virus by in situ hybridization using nonradioisotopic probes prepared by the polymerase chain reaction. Diagn. Mol. Pathol. 2 (2), 108–115. Varma, V. A.; Cerjan, C. M.; Abbott, K. L.; Hunter, S. B. (1994) Non-isotopic in situ hybridization method for mitochondria in oncocytes. J. Histochem. Cytochem. 42 (2), 273–276.

CONTENTS

INDEX

R eferences

B. RNA probes

C. Oligonucleotide probe

Brown, C. C.; Meyer, R. F.; Grubman, M. J. (1994) Identification of african horse sickness virus in cell culture using a digoxigenin-labeled RNA probe. J. Vet. Diagn. Invest. 6, 153–155.

b. 3'-Tailing

Coen, E. S.; Romero, J. M.; Doyle, S.; Elliott, R.; Murphy, G.; Carpenter, R. (1990) Floricaula: A homeotic gene required for flower development in Antirrhinum majus. Cell 63, 1311–1322. Marquardt, D. L.; Walker, L. L.; Heinemann, S. (1994) Cloning of two adenosine receptor subtypes from mouse bone marrow-derived mast cells. J. Immunol. 152, 4508–4515. Su, I.-J.; Chen, R.-L.; Lin, D.-T.; Lin, K.-S.; Chen, C.-C. (1994) Epstein-Barr virus (EBV) infects T lymphocytes in childhood EBV-associated hemophagocytic syndrome in Taiwan. Am. J. Pathol. 144 (6), 1219–1225.

Cubie, H. A.; Felix, D. H.; Southam, J. C.; Wray, D. (1991) Application of molecular techniques in the rapid diagnosis of EBV-associated oral hairy leukoplakia. J. Oral. Pathol. Med. 20, 271–274. Cubie, H. A.; Inglis, J. M.; McGowan, A. M. (1991) Detection of respiratory syncytial virus antigen and nucleic acid in clinical specimens using synthetic oligonucleotides. J. Virol. Meth. 34, 27–35. Oraveerakul, K.; Choi, C. S.; Molitor, T. W. (1993) Tissue tropisms of porcine parvovirus in swine. Arch. Virol. 130, 377–389. Pacchioni, D.; Papotti, M.; Bonio, F.; Bussolati, G.; Negro, F. (1992) Detection of cytomegalovirus by in situ hybridization using a digoxigenin-tailed oligonucleotide. Liver 12, 257–261.

c. Chemical synthesis Zischler, H.; Nanda, J.; Schäfer, R.; Schmid, M.; Epplen, J. (1989) Digoxigenated oligonucleotide probes specific for simple repeats in DNA fingerprinting and hybridization in situ. Hum. Genet. 82 (3), 227–233.

6

CONTENTS

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191

R eferences

3. Chromosomes/Interphase nuclei Antonacci, R.; Marzella, R.; Finelli, P.; Lonoce, A.; Forabosco, A.; Archidiacono, N.; Rocchi, M. (1995) A panel of subchromosomal painting libraries representing over 300 regions of the human genome. Cytogenet. Cell Genet. 68, 25–32.

Celeda, D.; Aldinger, K.; Haar, F.-M.; Hausmann, M.; Durm, M.; Ludwig, H.; Cremer, C. (1994) Rapid fluorescence in situ hybridization with repetitive DNA probes: Quantification by digital image analysis. Cytometry 17, 13–25.

Arnold, N.; Seibl, R.; Kessler, C.; Wienberg, J. (1992) Non-radioactive in situ hybridization with digoxigenin labeled DNA probes. Biotechnic Histochem. 67 (2), 59–67.

Chevillard, C.; Le Paslier, D.; Passage, E.; Ougen, P.; Billault, A.; Boyer, S.; Mazan, S.; Bachellerie, J. P.; Vignal, A.; Cohen, D.; Fontes, M. (1993) Relationship between Charcot-Marie-Tooth 1A and Smith-Magenis regions. snU3 may be a candidate gene for the Smith-Magenis syndrome. Hum. Mol. Gen. 2 (8), 1235–1243.

Arnoldus, E. P. J.; Wiegant, J.; Noordermeer, I. A.; Wessels, J. W.; Beerstock, G. C.; Grosveld, G. C.; van der Ploeg, M.; Raap, A. K. (1990) Detection of the Philadelphia chromosome in interphase nuclei. Cytogenet. Cell Genet. 54, 108–111. Ashley, T.; Ried, T.; Ward, D. C. (1994) Detection of nondisjunction and recombination in meiotic and postmeiotic cells from XYSxr [XY, Tp(Y)1Ct] mice using multicolor fluorescence in situ hybridization. Proc. Natl. Acad. Sci. USA 91, 524–528. Avarello, R.; Pedicini, A.; Caiulo, A.; Zuffardi, O.; Fraccaro, M. (1992) Evidence for an ancestral alphoid domain on the long arm of human chromosome 2. Hum. Genet 89, 247–249. Balazs, M.; Mayall, B. H.; Waldmann, F. M. (1991) Simultaneous analysis of chromosomal aneusomy and 5-bromodeoxyuridine incorporation in MCF-7 breast tumor cell line. Cancer Genet. Cytogenet. 57, 93–102. Boggs, B. A.; Chinault, C. (1994) Analysis of replication timing properties of human X-chromosomal loci by fluorescence in situ hybridization. Proc. Natl. Acad. Sci. USA 91, 6083–6087. Boschman, G. A.; Buys, C. H. C. M.; Van der Veen, A. Y.; Rens, W.; Osinga, J.; Slater, R. M.; Aten, J. A. (1993) Identification of a tumor marker chromosome by flow sorting, DNA amplification in vitro, and in situ hybridization of the amplified product. Genes Chrom. Cancer 6, 10–16.

6

Boyle, A. L.; Feltquite, D. M.; Dracopoli, N. C.; Housman, D. E.; Ward, D. C. (1992) Rapid physical mapping of cloned DNA on banded mouse chromosomes by fluorescence in situ hybridization. Genomics 12, 106–115. Brandt, C. A.; Hindkjaer, J.; Stromkjaer, H.; Pedersen, S.; Sunde, L.; Kolvraa, S. (1993) Molecular cytogenetics: Applications in clinical genetics. Europ. J. Obstetrics & Gynecol. Reprod. Biol. 50, 235–242. Brandt, C. A.; Lyngbye, T.; Pedersen, S.; Bolund, L.; Friedrich, U. (1994) Value of chromosome painting in determining the chromosomal outcome in offspring of a 12; 16 translocation carrier. J. Med. Genet. 31, 234–237. Celeda, D.; Bettag, U.; Cremer, C. (1992) PCR Amplification and simultaneous digoxigenin incorporation of long DNA probes for fluorescence in situ hybridization. BioTechn. 12 (1), 98–102.

192

Christmann, A.; Lagoda, P. J. L.; Zang, K. D. (1991) Non-radioactive in situ hybridization pattern of the M13 minisatellite sequences on human metaphase chromosomes. Hum. Genet. 86, 487–490. Couturier, J.; Garau-Sastre, X.; SchneiderMaunoury, S.; Labib, A.; Orth, G. (1991) Integration of papillomavirus DNA near myc genes in genital carcinomas and its consequences for protooncogene expression. J. Virol. Vol. 65, No. 8, 4534–4538. Dal Cin, P.; Aly, M. S.; Delabie, J.; Ceuppens, J. L.; Van Gool, S.; Van Damme, B.; Baert, L.; Van Poppel, H.; Van Den Berghe, H. (1992) Trisomy 7 and trisomy 10 characterize subpopulations of tumorinfiltrating lymphocytes in kidney tumors and in the surrounding kidney tissue. Proc. Natl. Acad. Sci. USA 89, 9744–9748. Dauwerse, J. G.; Wiegant, J.; Raap, A. K.; Breuning, M. H.; Ommen, van, G. J. B. (1992) Multiple colors by fluorescence in situ hybridization using ratiolabeled DNA probes create a molecular karyotype. Hum. Mol. Gen. 1 (8), 593–598. Davies, A. F.; Barber, L.; Murer-Orlando, M.; Bobrow, M.; Adinolfi, M. (1994) FISH detection of trisomy 21 in interphase by the simultaneous use of two differentially labeled cosmid contigs. J. Med. Genet. 31, 679–685. De Frutos, R.; Kimura, K.; Peterson, K. R. (1990) In situ hybridization of Drosophila polytene chromosomes with digoxigenin-dUTP labeled probes. Methods Mol. Cell. Biol. 2, 32–36. Delhanty, J. D. A.; Griffin, D. K.; Handyside, A. H.; Harper, J.; Atkinson, G. H. G.; Pieters, M. H. E. C.; Winston, R. M. L. (1993) Detection of aneuploidy and chromosomal mosaicism in human embryos during preimplantation sex determination by fluorescent in situ hybridzation (FISH). Hum. Mol. Gen. 2 (8), 1183–1185. De Leeuw, B.; Suijkerbuijk, R. F.; Balemans, M.; Sinke, R. J.; De Jong, B.; Molenaar, W. M.; Meloni, A. M.; Sandberg, A. A.; Geraghty, M.; Hofker, M.; Ropers, H. H.; Geurts van Kessel, A. (1993) Sublocalization of the synovial sarcoma-associated t(X;18) chromosomal breakpoint in Xp11.2 using cosmid cloning and fluorescence in situ hybridization. Oncogene 8, 1457–1463.

CONTENTS

INDEX

R eferences

De Leeuw, B.; Berger, W.; Sinke, R. J.; Suijkerbuijk, R. F.; Gilgenkrantz, S.; Geraghty, M. T.; Valle, D.; Monaco, A. P.; Lehrach, H.; Ropers, H. H.; Geurts van Kessel, A. (1993) Identification of a yeast artificial chromosome (YAC) spanning the synovial sarcoma-specific t(X;18)(p11.2;q11.2) Breakpoint. Genes, Chrom. Cancer 6, 182–189. Desmaze, C.; Zucman, J.; Delattre, O.; Thomas, G.; Aurias, A. (1992) In situ hybridization of PCR amplified inter-Alu sequences from a hybrid cell line. Hum. Genet. 88, 541–544. De Vries, J. E.; Kornips, F. H. A. C.; Wiegant, J.; Moerkerk, P. M.; Senden, N.; Schutte, B.; Geraedts, J. P. M.; Bosman, F. T.; ten Kate, J. (1992) Chromosomal localization of transfected genes by a combination of hot banding and fluorescence in situ hybridization. Döhner, H.; Pohl, S.; Bulgay-Mörschel, M.; Stilgenbauer, S.; Bentz, M.; Lichter, P. (1993) Trisomy 12 in Chronic Lymphoid Leukemias – a Metaphase and Interphase Cytogenetic Analysis. Leukemia 7 (4), 516–520. Dunham, I.; Lengauer, C.; Cremer, T.; Featherstone, T. (1992) Rapid generation of chromosome-specific alphoid DNA probes using the polymerase chain reaction. Hum. Genet. 88, 457–462. Fidlerová, H.; Senger, G.; Kost, M.; Sanseau, P.; Sheer, D (1994) Two simple procedures for releasing chromatin from routinely fixed cells for fluorescence in situ hybridization. Cytogenet. Cell Genet. 65, 203–205. Flejter, W. L.; Barcroft, C. L.; Guo, S.-W.; Lynch, E. D.; Boehnke, M.; Chandrasekharappa, S.; Hayes, S.; Collins, F. S.; Weber, B. L.; Glover, T. W. (1993) Multicolor FISH mapping with Alu-PCR-Amplified YAC Clone DNA determines the order of markers in the BRCA1 region on chromosome 17q12-q21. Genomics 17, 624–631. Gerdes, M. G.; Carter, K. C.; Moen, P. T.; Lawrence, J. B. (1994) Dynamic changes in the higher-level chromatin organization of specific sequences revealed by in situ hybridization to nuclear halos. J. Cell Biol. 126 (2), 289–304. Geurts van Kessel, A.; Stellink, F.; van Gaal, J.; van de Klundert, W.; Siepman, A.; Oosten, H. R. (1994) Translocation (12;22) (p13;q12) as sole karyotypic abnormality in a patient with nonlymphocytic leukemia. Cancer Genet. Cytogenet. 72, 105–108. Gray, J. W.; Pinkel, D.; Brown, J. M. (1994) Fluorescence in situ hybridization in cancer and radiation biology. Radiat. Res. 137, 275–289. Haar, F.-M.; Durm, M.; Aldinger, K.; Celeda, D.; Hausmann, M.; Ludwig, H.; Cremer, C. (1994) A rapid FISH technique for quantitative microscopy. BioTechn. 17 (2), 346–355. Han, T. L.; Ford, J. H.; Webb, G. C.; Flaherty, S. P.; Correll, A.; Matthews, C. D. (1993) Simultaneous detection of X- and Y-bearing human sperm by double fluorescence in situ hybridization. Mol. Reprod. Develop. 34, 308–313.

CONTENTS

INDEX

Han, T. L.; Ford, J. H.; Flaherty, S. P.; Webb, G. C.; Matthews, C. D. (1994) A fluorescent in situ hybridization analysis of the chromosome constitution of ejaculated sperm in a 47,XYY male. Clin. Genet. 45, 57–70. Hausmann, M.; Dudin, G.; Aten, J. A.; Heilig, R.; Diaz, E.; Cremer, C. (1991) Slit Scan Flow Cytometry of isolated chromosomes following fluorescence hybridization: an Approach of online screening for specific chromosomes and chromosome translocations. Z. Naturforsch. 46c, 433–441. Heppell-Parton, A. C.; Albertson, D. G.; Fishpool, R.; Rabbitts, P. H. (1994) Multicolour fluorescence in situ hybridization to order small, single-copy probes on metaphase chromosomes. Cytogenet. Cell Genet. 66, 42–47. Heslop-Harrison, J. S.; Schwarzacher, T.; Anamthawat-Jonsson, K.; Leitch, A. R.; Shi, M.; Leitch, I. J. (1991) In situ hybridization with automated chromosome denaturation. Technique 3 (3), 109–116. Hyytinen, E.; Visakorpi, T.; Kallioniemi, A.; Kallioniemi, O.-P.; Isola, J. J. (1994) Improved technique for analysis of formalin-fixed, paraffinembedded tumors by fluorescence in situ hybridization. Cytometry 16, 93–99. Jansen, G.; Bartolomei, M.; Kalscheuer, V.; Merkx, G.; Wormskamp, N.; Mariman, E.; Smeets, D.; Ropers, H.-H.; Wieringa, B. (1993) No imprinting involved in the expression of DM-kinase mRNAs in mouse and human tissues. Hum. Mol. Gen. 2 (8), 1221–1227. Jauch, A.; Wienberg, J.; Stanyon, R.; Arnold, N.; Tofanelli, S.; Ishida, T.; Cremer, T. (1992) Reconstruction of genomic rearrangements in great apes and gibbons by chromosome painting. Proc. Natl. Acad. Sci. USA 89, 8611–8615. Jenne, D. E.; Zimmer, M.; Garcia-Sanz, J. A.; Tschopp, J.; Lichter, P. (1991) Genomic organization and subchromosomal in situ localization of the murine granzyme F, a serine protease expressed in CD8+ T cells. J. Immunol. 147, 1045–1052.

6

Johnson, C. V.; McNeil, J.; Carter, K. C.; Lawrence, J. B. (1991) A simple, rapid technique for precise mapping of multiple sequences in two colors using a single optical filter set. GATA 8 (2), 75–76. Joos, S.; Scherthan, H.; Speicher, M. R.; Schlegel, J.; Cremer, T.; Lichter, P. (1993) Detection of amplified DNA sequences by reverse chromosome painting using genomic tumor DNA as probe. Hum. Genet. 90, 584–589. Kallioniemi, A.; Kallinoniemi, O.-P.; Piper, J.; Tanner, M.; Stokke, T.; Chen, L.; Smith, H. S.; Pinkel, D.; Gray, J. W.; Waldman, F. M. (1994) Detection and mapping of amplified DNA sequences in breast cancer by comparative genomic hybridization. Proc. Natl. Acad. Sci. USA 91, 2156–2160. Kallioniemi, O.-P.; Kallioniemi, A.; Piper, J.; Isola, J.; Waldman, F. M.; Gray, J. W.; Pinkel, D. (1994) Optimizing comparative genomic hybridization for analysis of DNA sequence copy number changes in solid tumors. Genes, Chrom. Canc. 10, 231–243.

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Lawrence, J. B.; Singer, R. H.; McNeil, J. A. (1990) Interphase and metaphase resolution of different distances within the human dystrophin gene. Science 249, 928–932. Lazaridou, A.; Chase, A.; Melo, J.; Garicochea, B.; Diamond, J.; Goldman, J. (1994) Lack of reciprocal translocation in BCR-ABL positive Ph-negative chronic myeloid leukaemia. Leukemia 8 (3), 454–457. Le Beau, M. M.; Espinosa III, R.; Neuman, W. L.; Stock, W.; Roulston, D.; Larson, R. A.; Keinanen, M.; Westbrook, C. A. (1993) Cytogenetic and molecular delineation of the smallest commonly deleted region of chromosome 5 in malignant myeloid diseases. Proc. Natl. Acad. Sci. USA 90, 5484–5488. Léger, I.; Thomas, M.; Ronot, X.; Brugal, G. (1993) Detection of chromosomes 1 aberrations by fluorescent in situ hybridization (FISH) in the human breast cancer cell line MCF-7. Anal. Cell. Pathol. 5, 299–309. Léger, I.; Robert-Nicoud, M.; Brugal, G. (1994) Combination of DNA in situ hybridization and immunocytochemical detection of nucleolar proteins: A contribution to the functional mapping of the human genome by fluorescence microscopy. J. Histochem. Cytochem. 42 (2), 149–154. Léger, I.; Guillaud, M.; Krief, B.; Brugal, G. (1994) Interactive computer-assisted analysis of chromosome 1 colocalization with nucleoli. Cytometry 16, 313–323. Leitch, A. R.; Mosgöller, W.; Shi, M.; HeslopHarrison, J. S. (1992) Different patterns of rDNA organization at interphase in nuclei of wheat and rye. J. Cell Sci. 101, 751–757.

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Pendás, A. M.; Morán, P.; Garcia-Vázquez, E. (1993) Ribosomal RNA genes are interspersed throughout a heterochromatic chromosome arm in Atlantic salmon. Cytogenet. Cell Genet. 63, 128–130. Pendás, A. M.; Morán, P.; Garcia-Vázquez, E. (1993) Multi-chromosomal location of ribosomal RNA genes and heterochromatin association in brown trout. Chrom. Res. 1, 63–67. Popp, S.; Jauch, A.; Schindler, D.; Speicher, M. R.; Lengauer, C.; Donis-Keller, H.; Riethman, H. C.; Cremer, T. (1993) A strategy for the characterization of minute chromosome rearrangements using multiple color fluorescence in situ hybridization with chromosome-specific DNA libraries and YAC clones. Hum. Genet. 92, 527–532. Raap, A. K.; Wiegant, J.; Lichter, P. Multiple fluorescence in situ hybridization for molecular cytogenetics. Rassool, F. V.; McKeithan, T. W.; Neilly, M. E.; Van Melle, E.; Espinosa III, R.; Le Beau, M. M. (1991) Preferential integration of marker DNA into the chromosomal fragile site at 3p14: an approach to cloning fragile sites. Proc. Natl. Acad. Sci. USA 88, 6657–6661. Richard, F.; Vogt, N.; Muleris, M.; Malfoy, B.; Dutrillaux, B. (1994) Increased FISH efficieny using APC probes generated by direct incorporation of labeled nucleotides by PCR. Cytogenet. Cell Genet. 65, 169–171. Ried, T.; Arnold, N.; Ward, D. C.; Wienberg, J. (1993) Comparative high-resolution mapping of human and primate chromosomes by fluorescence in situ hybridization. Genomics 18, 381–386. Ried, T.; Baldini, A.; Rand, T. C.; Ward, D. C. (1992) Simultaneous visualization of seven different DNA probes by in situ hybridization using combinatorial fluorescence and digital imaging microscopy. Proc. Natl. Acad. Sci. USA 89, 1388–1392. Ried, T.; Landes, G.; Dackowski, W.; Klinger, K.; Ward, D. C. (1992) Multicolor fluorescence in situ hybridization for the simultaneous detection of probe sets for chromosomes 13, 18, 21, X and Y in uncultured amniotic fluid cells. Human Mol. Gen. 1 (5), 307–313.

6

Ried, T.; Lengauer, C.; Cremer, T.; Wiegant, J.; Raap, A. K.; van der Ploeg, M.; Groitl, P.; Lipp, M. (1991) Specific metaphase and interphase detection of the breakpoint region in 8q24 of burkitt lymphoma cells by triple-color fluorescence in situ hybridization. Genes, Chromosomes & Cancer 4, 69–74. Saitoh, Y.; Mizuno, S. (1992) Distribution of Xho I and Eco RI family repetitive DNA sequences into separate domains in the chicken W chromosome. Chromosoma 101, 474–477. Scherthan, H.; Köhler, M.; Vogt, P.; Malsch, von, K.; Schweizer, D. (1992) Chromosomal in situ hybridization with double-labeled DNA: signal amplification at the probe level. Cytogenet. Cell Genet. 60, 4–7.

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Speleman, F.; Leroy, J. G.; Roy, van, N.; De Paepe, A.; Suijkerbuijk, R.; Brunner, H.; Looijenga, L.; Verschraegen-Spae, M.-R.; Orye, E. (1991) Pallister-Killian syndrome: Characterization of the isochromosome 12p by fluorescent in situ hybridization. Am. J. Med. Gen. 41, 381–387. Speleman, F.; Van Roy, N.; De Vos, E.; Hilliker, C.; Suijkerbuijk, R. F. S.; Leroy, J.G. (1993) Molecular cytogenetic analysis of a familial pericentric inversion of chromosome 12. Clin. Genet. 44, 156–163. Spriggs, E. L.; Martin, R. H. (1994) Analysis of segregation in a human male reciprocal translocation carrier, t (1;11) (p36.3;q13.1), by twocolour fluorescence in situ hybridization. Mol. Reprod. Develop. 38, 247–250.

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Wiegant, J.; Ried, T.; Nederlof, P. M.; van der Ploeg, M.; Tanke, H. J. Raap, A. K. (1991) In situ hybridization with fluoresceinated DNA. Nucl. Acids Res. 19 (12), 3237–3241. Wiegant, J.; Wiesmeijer, C. C.; Hoovers, J. M. N.; Schuuring, E.; D’Azzo, A.; Vrolijk, J.; Tanke, H. J.; Raap, A. K. (1993) Multiple and sensitive fluorescence in situ hybridization with rhodamine-, fluorescein-, and coumarin-labeled DNAs. Cytogenet. Cell Genet. 63, 73–76. Wienberg, J.; Stanyon, R.; Jauch, A.; Cremer, T. (1992) Homologies in human and Macaca fuscata chromosomes revealed by in situ suppression hybridization with human chromosome specific DNA libraries. Chromosoma 101, 265–270. Wyrobek, A. J.; Robbins, W. A.; Mehraein, Y.; Pinkel, D.; Weier, H.-U. (1994) Detection of sex chromosomal aneuploidies X-X, Y-Y, and X-Y in human sperm using two-chromosome fluroescence in situ hybridization. Am. J. Med. Gen. 53, 1–7. Yerle, M.; Goureau, A.; Gellin, J.; Le Tissier, P.; Moran, C. (1994) Rapid mapping of cosmid clones on pig chromosomes by fluorescence in situ hybridization. Mammalian Genome 5, 34–37. Zhang, F. R.; Heilig, R.; Thomas, G.; Aurias, A. (1990) A one-step efficient and specific nonradioactive non-fluorescent method for in situ hybridization of banded chromosomes. Chromosoma 99, 436–439.

6

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197

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4. Primed in Situ Labeling of Nucleic Acids Abbo, S.; Dunford, R. P.; Miller, T. E.; Reader, S. M.; King, I. P. (1993) Primer-mediated in situ detection of the B-hordein gene cluster on barley chromosome 1H. Proc. Natl. Acad. Sci. USA 90, 11821–11824. Brandt, C. A.; Djernes, B.; Stromkjaer, H.; Petersen, M. B.; Pedersen, S.; Hindkjaer, J.; Brinch-Iversen, J.; Bruun-Petersen, G. (1994) Pseudododicentric chromosome 18 diagnosed by chromosome painting and primed in situ labeling (PRINS). J. Med. Genet. 31, 99–102. Brandt, C. A.; Kierkegaard, O.; Hindkjaer, J.; Jensen, P. K. A.; Pedersen, S.; Therkelsen, A. J. (1993) Ring chromosome 20 with loss of telomeric sequences detected by multicolour PRINS. Clin. Genet. 44, 26–31. Cinti, C.; Santi, S.; Maraldi, N. M. (1993) Localization of single copy gene by PRINS technique. Nucl. Acids Res. 21 (24), 5799–5800. Gosden, J.; Hanratty, D. (1991) Comparison of sensitivity of three haptens in the PRINS (oligonucleotide primed in situ DNA synthesis) reaction. Techniques 3 (4), 159–165. Gosden, J.; Lawson, D. (1995) Instant PRINS: a rapid method for chromosome identification by detecting repeated sequences in situ. Cytogenet. Cell Genet. 68, 57–60. Hindkjaer, J.; Koch, J.; Mogensen, J.; Pedersen, S.; Fischer, H.; Nygaard, M.; Junker, S.; Gregersen, N.; Kolvraa, S.; Therkelsen, A. J.; Bolund, L. (1991) Primed in situ labeling of nucleic acids. BFE 8, (12) 752–756. Hindkjaer, J.; Koch, J.; Terkelsen, C.; Brandt, C. A.; Kolvraa, S.; Bolund, L. (1994) Fast, sensitive multicolor detection of nucleic acids in situ by PRimed IN Situ labeling (PRINS). Cytogenet. Cell Genet. 66, 152–154.

6

Koch, J.; Fischer, H.; Askholm, H.; Hindkjaer, J.; Pedersen, S.; Kolvraa, S.; Bolund, L. (1993) Identification of a supernumerary der(18) chromosome by a rational strategy for the cytogenetic typing of small marker chromosomes with chromosome-specific DNA probes. Clin. Genet. 43, 200–203. Koch, J.; Hindkjaer, J.; Kolvraa, S.; Bolund, L. Construction of a panel of chromosome specific oligonucleotide probes (PRINS-primers) useful for the identification of individual human chromosomes in situ. Cytogen. Cell Gen. submitted. Pellestor, F.; Girardet, A.; Andréo, B.; Charlieu, J.-P. (1994) A polymorphic alpha satellite sequence for human chromosome 13 detected by oligonucleotide primed in situ labeling (PRINS). Hum. Genet. 94, 346–348. Pellestor, F.; Girardet, A.; Geneviève, L.; Andréo, B.; Charlieu, J. P. (1995) Use of the primed in situ labeling (PRINS) technique for a rapid detection of chromosomes 13, 16, 18, 21, X and Y. Hum. Genet. 95, 12–17. Speel, E. J. M.; Lawson, D.; Hopman, A. H. N.; Gosden, J. (1995) Multi-PRINS: multiple sequential oligonucleotide primed in situ DNA synthesis reactions label specific chromosomes and produce bands. Hum. Genet. 95, 29–33. Terkelsen, C.; Koch, J.; Kolvraa, S.; Hindkjaer, J.; Pedersen, S.; Bolund, L. (1993) Repeated primed in situ labeling: formation and labeling of specific DNA sequences in chromosomes and nucleic. Cytogenet. Cell Genet. 63, 235–237. Therkelsen, A. J.; Nielsen, A.; Koch, J.; Hindkjaer, J.; Kolvraa, S. (1995) Staining of human telomeres with primed in situ labeling (PRINS). Cytogenet. Cell Genet. 68, 115–118. Volpi, E. V.; Baldini, A. (1993) Multiprins: a method for multicolour primed in situ labeling. Chrom. Res. 1, 257–260.

Hindkjaer, J.; Koch, J.; Mogensen, J.; Kolvraa, S.; Bolund, L. (1994) Primed in situ (PRINS) labeling of DNA. Meth. Mol. Biol. 33, 95–109.

5. In situ hybridization in suspension Brown, C. C.; Meyer, R. F.; Grubman, M. J. (1993) Use of digoxigenin-labeled RNA probe to detect all 24 serotypes of bluetongue virus in cell culture. J. Vet. Diagn. Invest. 5, 159–162. Timm, A.; Stewart, C. C. (1992) Fluorescent in situ hybridization en suspension (FISHES) using digoxigenin-labeled probes and flow cytometry. BioTechn. 12 (3), 362–367.

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6. PCR in situ technique Cinti, C.; Santi, S.; Maraldi, N. M. (1993) Localization of single copy gene by PRINS technique. Nucl. Acids Res. 21 (24), 5799–5800. Gosden, J.; Hanratty, D. (1993) PCR In situ: A rapid alternative to in situ hybridization for mapping short, low copy number sequences without isotopes. BioTechn. 15 (1), 78–80. Gressens, P.; Martin, J. R. (1994) In situ polymerase chain reaction: localization of HSV-2 DNA sequences in infections of the nervous system. J. Virol. Meth. 46, 61–83. Li, S.; Harris, C. P.; Leong, S. A. (1993) Comparison of fluorescence in situ hybridization and primed in situ labeling methods for detection of single-copy genes in the fungus ustilago maydis. Experim. Mycol. 17, 301–308. Long, A. A.; Komminoth, P.; Lee, E.; Wolfe, H. J. (1993) Comparison of indirect and direct in situ polymerase chain reaction in cell preparations and tissue sections. Histochem. 99, 151–162. Moss, R. B.; Kaliner, M. A. (1994) Primed in Situ DNA Amplification (PIDA). J. Clin. Lab. Anal. 8, 120–122. Nuovo, G.; Direct incorporation of digoxigenin-11dUTP into amplified DNA using the “ Hot Start” PCR in situ technique. Nuovo, G. J.; Gallery, F.; MacConnell, P.; Becker, J.; Bloch, W. (1991) An improved technique for the in situ detection of DNA after Polymerase Chain Reaction amplification. Am. J. Pathol. 139 (6), 1239–1244.

Patel, V. G.; Shum-Siu, A.; Heniford, B. W.; Wieman, T. J.; Hendler, F. J. (1994) Detection of epidermal growth factor receptor mRNA in tissue sections from biopsy specimens using in situ polymerase chain reaction. Am. J. Pathol. 144 (1), 7–14. Pellestor, F.; Girardet, A.; Lefort, G.; Andréo, B.; Charlieu, J. P. (1994) Détection rapide des chromosomes 21 in situ par la technqie PRINS. Ann. Génét. 37 (2), 66–71. Pestaner, J. P.; Bibbo, M.; Bobroski, L.; Seshamma, T.; Bagasra, O. (1994) Potential of the in situ polymerase chain reaction in diagnostic cytology. Acta Cytol. 38, 676–680. Teyssier, M.; Carosella, E.; Gluckman, E.; Kirszenbau, M. (1994) PCR introcellulaire: nouvelle approche de diagnostic tissulaire et cytogenetique. Immunoanal. Biol. Spec. 9, 159–164. Tsongalis, G. J.; McPhail, A. H.; Lodge-Rigal, R. D.; Chapman, J. F.; Silverman, L. M. (1994) Localized in situ amplification (LISA): A novel approach to in situ PCR. Clin. Chem. 40 (3), 381–384. Zehbe, I.; Hacker, G. W.; Rylander, E.; Sällström, J.; Wilander, E. (1992) Detection of single HPV copies in SiHa cells by in situ polymerase chain reaction (in situ PCR) combined with immuno-peroxidase and immunogold-silver staining (IGSS) techniques. Anticanc. Res. 12, 2165–2168.

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INDEX

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