Short Technical Reports Fluorescent in situ hybridization employing ...

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1California Institute of Technology, Pasadena, CA and 2The University of ... 1000 Sectioning System from Technical. Products ..... by the NSF CAREER grant no.
Short Technical Reports Fluorescent in situ hybridization employing the conventional NBT/BCIP chromogenic stain Le A. Trinh1, Marshall D. McCutchen2, Marianne Bonner-Fraser1, Scott E. Fraser1, Lloyd A. Bumm2, and David W. McCauley1,2 1California

Institute of Technology, Pasadena, CA and 2The University of Oklahoma, Norman, OK, USA

BioTechniques 42:756-759 (June 2007) doi 10.2144/000112476

In situ hybridization techniques typically employ chromogenic staining by enzymatic amplification to detect domains of gene expression. We demonstrate the previously unreported near infrared (NIR) fluorescence of the dark purple stain formed from the commonly used chromogens, nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP). The solid reaction product has significant fluorescence that enables the use of confocal microscopy to generate high-resolution three-dimensional (3-D) imaging of gene expression.

INTRODUCTION In situ hybridization techniques using labeled nucleic acid probes have revolutionized our ability to visualize the presence and distribution of RNA transcripts within fixed tissues and cells (1,2). Early approaches employed radiolabeled probes, but more recent nonradioactive methods for detecting mRNA expression patterns involve both chromogenic stains and fluorescent dyes. The typical chromogenic method of detecting mRNA expression involves the incorporation of the plant steroid digoxigenin (DIG) into an RNA probe (3,4). After annealing with the endogenous RNA, the labeled probe is detected by immunolabeling with an antibody to DIG coupled to the alkaline phosphatase (AP) enzyme. The conventional chromogenic method for detecting AP activity is a combination of nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) called NBT/BCIP (5). In this method, the hydrolysis of BCIP by AP results in the indoxyl that dimerizes to form the leucoindigo, which is in turn oxidized by NBT to the insoluble blue 5,5′-dibromo-4,4′dichloro indigo (BCI). In this reaction, NBT is reduced to the insoluble purple diformazan (DF), making NBT-DF (6,7). Thus, this reaction produces an insoluble dark purple precipitate that is a mixture of NBT-DF and BCI. This 756 ı BioTechniques ı www.biotechniques.com

chromogenic method is very sensitive due to enzymatic amplification, because one AP can hydrolyze many BCIP molecules. The result is a stain that can be visualized easily by light microscopy, but it is often difficult to determine the three-dimensional (3-D) pattern of expression without advanced imaging tools such as optical projection microscopy (8). Fluorescence methods offer the advantage of being able to determine RNA expression in three dimensions when combined with optical sectioning techniques such as confocal laser scanning microscopy (CLSM). This added depth discrimination yields greater resolution of transcripts and the ability to place RNA expression into the context of the surrounding tissues and structures. In recent years, efforts have advanced toward refining fluorescent in situ hybridization techniques in two avenues: (i) to develop approaches that generate more signal per transcript to make up for the significant loss of signal by conventional immunofluorescence approaches; and (ii) to refine multiplex approaches that permit the detection of multiple RNAs within a single sample (9). In systems where both developments have been refined, fluorescence approaches have facilitated the identification of molecular signatures in individual cells; however, they are difficult to optimize and have

a number of limitations, including high background, low signal-to-noise ratio, and instability of the signal once staining is completed. For these reasons, the standard chromogenic methods for visualizing the presence of mRNAs remain dominant. Here, we report that the advantages of fluorescence in situ hybridization are easily available, as the widely used NBT/BCIP chromogenic stain is a fluorophore, a fact that had previously gone unrecognized. MATERIALS AND METHODS Two imaging systems were used for the excitation and detection of the NBT/BCIP stain. The first, a Zeiss LSM 510 laser scanning confocal microscope with a 633-nm helium-neon (HeNe) laser and a 650-nm long pass emission filter, was used for detection of the NBT/BCIP signal. The long pass filter collects all emitted light above 650 nm. The second was a Zeiss Axio Imager Z1 system with an Hg arc lamp source, a 645–685 nm excitation band pass filter, and a 760-nm long pass emission filter (Chroma Technology; www.chroma.com) coupled to an Ocean Optics HR2000 charged-couple device (CCD) spectrometer via a 200μm core optical fiber mounted at the focal plane of the camera port that was used to determine the emission spectra. The optical fiber only collects the light falling on its core, which for the Zeiss EC Plan Neofluar® 40×/1.3 numerical aperture (NA) objective used in this work defines a 5-μm diameter area of the sample. Spatial correlation between the image and the spectra (fiber core) was established using an x-y translator in the optical fiber mount to align it to the eyepiece crosshair reticle. The NBT/BCIP staining protocol was performed as follows: zebrafish and lamprey whole-mount embryos incubated with DIG-labeled riboprobes were immunolabeled with a sheep antiDIG antibody conjugated to AP (1:3000; Roche Diagnostics, Indianapolis, IN, USA). They were subsequently processed with NBT/BCIP solution (Roche Diagnostics) according to the manufacturer’s instructions to obtain the dark purple NBT-DF/BCI stain. Immunohistochemistry following Vol. 42 ı No. 6 ı 2007

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Figure 1. Fluorescent imaging of in situ hybridization stained with nitro blue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP). (A–C) Expression of muscle actin2 (MA2) vibratome sectioned (100 μm) through branchial arches of a stage-25 lamprey embryo. (A) Lamprey autofluorescence from excitation at 488 nm is quenched in the regions containing the NBT-diformazan (DF)/5,5′-dibromo-4,4′-dichloro indigo (BCI) precipitate following in situ hybridization with a MA2 riboprobe, while outlining the cells and lipid droplets of the arches where the precipitate is absent. (B) Fluorescence of NBTDF/BCI precipitate upon excitation at 633 nm. (C) Merged image from panels A and B. (D–E) Transverse 200-μm vibratome section of cardiac myosin light chain2 (cmlc2) expression in 20-somite stage zebrafish embryo; dorsal to the top. (D) Transmitted light [differential interference contrast (DIC)] image of cmlc2 expression in the myocardial precursors of a zebrafish embryo as visualized by the bluish-purple NBT-DF/BCI precipitates. (E) Laser scanning confocal microscope image of the NBT-DF/BCI precipitates in panel A captured at a single Z-plane. The NBT-DF/BCI precipitates were excited with a 633-nm laser and detected with a 650-nm long pass filter on a Zeiss LSM 510 laser scanning confocal microscope; image was counterstained with an antibody against β-catenin (green). (F) Projected dorsal view of a 52-μm Z-stack of tg(flk1::eGFP) embryo stained with NBT/BCIP by in situ hybridization for cmlc2 (red) and immunohistochemistry for green fluorescent protein (GFP) (green) at 17-somite; anterior to top. Scale bars, 20 μm.

NBT/BCIP staining was performed as follows: anti-green fluorescent protein (GFP) antibody (1:500; Torrey Pines Biolabs, Houston, TX, USA) and antiβ-catenin (1:500; Sigma-Aldrich, St. Louis, MO, USA) were incubated with embryos overnight at 4°C, followed by incubation with an anti-rabbit or antimouse Alexa-conjugated secondary (1:200; Molecular Probes™; Invitrogen, Carlsbad, CA, USA). Stained embryos were embedded in 4% NuSieve® GTG low melting agarose (Fisher Scientific, Pittsburgh, PA, USA) and cut into 100or 200-μm sections with a Vibratome® 1000 Sectioning System from Technical Products International (St. Louis, MO, USA) for imaging. For a projected view of wholemount samples (Figure 1F), embryos were embedded in 4% low melting agarose with the dorsal side of the embryo positioned parallel to the plane of imaging. Z-slices were imaged as described previously at 1.5-μm intervals. A subset of the Z-slices that contained the stained tissue was then projected along the y-axis using Zeiss LSM software. NBT-DF was prepared by dissolving 20 mg NBT dichloride in 25 mL Vol. 42 ı No. 6 ı 2007

absolute ethanol. A solution of sodium borohydride was added with stirring (100 mg in 10 mL absolute ethanol). A deep purple precipitate formed and was allowed to stand overnight. The precipitate was filtered, washed with absolute ethanol, then washed with acetone. The acetone wash was continued until the wash was clear (approximately 30 mL) to remove the monoformazan. The resulting solid was bluish-black. The precipitate was soluble in nitrobenzene, and crystallization from nitrobenzene gave purple needles. The visible/near infrared (VIS/NIR) absorption spectra of the solution of this product in nitrobenzene is consistent with previously reported spectra (10,11) (data not shown). RESULTS AND DISCUSSION We initially noticed that the NBT/ BCIP chromogenic stain exhibits advantageous optical properties when attempting to assess the autofluorescence of tissues in lamprey embryos (12). Autofluorescence of tissues in lamprey embryos has posed a major obstacle to applying fluorescent

techniques to this organism. We reasoned that this autofluorescence could be turned into an advantage by employing the strong optical absorption of the NBT/BCIP stain to produce a negative fluorescence signal. This regional loss of autofluorescence could be reconstructed in three dimensions by CLSM, yielding a cellular resolution image of gene expression. As expected, when the sample was excited with the 488-nm laser, and 510–530 nm emitted light was collected, lamprey embryos stained for expression of muscle actin2 (MA2) revealed a negative signal in the region of the forming branchial arches that were strongly positive for the NBT/ BCIP stain (Figure 1A, arrowheads). To our surprise, the NBT/BCIP stain could be detected directly as an intense signal when excited with a 633-nm laser, and the emitted light was collected with a 650-nm long pass filter (Figure 1, B and C, arrows). The emission of light from the NBT/BCIP stain at a wavelength longer than the excitation wavelength suggested that this was a fluorescent signal. To demonstrate the broad applicability of the fluorescence of the www.biotechniques.com ı BioTechniques ı 757

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NBT/BCIP staining, we assessed imaging mRNA expression in multiple organisms. A standard confocal microscope (Zeiss LSM 510) can capture the fluorescence signal from the stain after whole-mount in situ hybridization in zebrafish embryos (Figure 1, E and F). No special procedure was needed; whole-mount in situ hybridizations were performed as previously described (13,14). The fluorescence of the NBT/BCIP precipitate offers

significantly improved resolution of mRNA distribution over the resolution obtained by conventional transmitted light microscopy. Light microscopy of transverse tissue sections of the zebrafish embryo can place the domains where the cardiac myosin light chain2 (cmlc2) gene is expressed into their proper context (Figure 1D). In contrast, CLSM of whole-mounted embryos stained with NBT/BCIP offers higher resolution and better contrast (Figure

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Figure 2. Nitro blue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) staining generates a bright and stable fluorescent precipitate. (A) Fluorescence spectra of the NBT/BCIPstained tissue (magenta), NBT-diformazan (DF) crystals (orange), and blue 5,5′-dibromo-4,4′-dichloro indigo (BCI)-stained tissue (blue), as well as background autofluorescence and scattering from unstained tissue (green). The spectrum obtained with a mirror in the sample position (red) shows the worst-case spectral leakage of the filter set. All measurements were taken with the same illumination intensity and excited with an Hg arc lamp and a filter that allows light between 645–685 nm to pass to the sample. Inset: fluorescence image of the NBT-DF needles. (B) Photostability of the NBT/BCIP stain under constant illumination. The maximum illumination intensity (HBO 100 Hg bulb) was used, and fluorescence emission is integrated over the band of 700–1100 nm. The fluorescence intensity falls to half its initial value in 27 min under these conditions. Insets: transmitted light (differential interference contrast) images of stained tissue before (above) and after (below) bleaching. 758 ı BioTechniques ı www.biotechniques.com

1E). The expression of cmlc2 in a subset of cells within the developing heart field can be resolved to individual cmlc2-expressing cells, as the stain is excluded from cell nuclei (Figure 1, E and F, yellow arrows). Similar highresolution images have been obtained from chick, frog, and amphioxus tissue mounted in agarose or glycerol (data not shown). By combining in situ hybridization with antibody staining, we are able to assess the relative positioning of cells expressing different markers during development. Figure 1F shows an example of in situ hybridization with the cmlc2 RNA probe in which endothelial precursors have been revealed by antibody staining of GFP in transgenic embryos [tg(flk1::eGFP)] (15). The endothelial precursors (GFPpositive) are both medial and dorsal to the myocardial precursors (cmlc2positive), providing a clear discrimination of the relative positioning of the two cell types that give rise to the heart tube during their morphogenetic movements to the midline. The reaction products of the NBT/ BCIP stain (NBT-DF and BCI) are not known fluorophores; therefore, we characterized their optical emission spectra. Figure 2A shows the emission spectra of stained (magenta curve), and adjacent unstained (green curve; shown 10× larger scale for clarity) lamprey tissue expressing MA2 (Figure 2B, upper inset). These spectra show that the emission from the stained region in the embryo is red-shifted from the excitation light (645–685 nm), as would be expected for fluorescence, and show a dual peak with maxima at 823 and 855 nm (Figure 2A, asterisks). To test the individual components of the NBT/BCIP stain for fluorescence, we stained lamprey embryos with BCIP only and reduced NBT with sodium borohydride to generate NBTDF crystals. Fluorescence emission of BCIP after hydrolysis by AP to form BCI was observed to peak at 770 nm (Figure 2A, double dagger). While BCI fluoresces, it is highly diffusible in the absence of NBT-DF and does not remain localized at the sight of gene expression; therefore by itself, it is not suitable for high-resolution imaging (data not shown). In contrast, pure NBTVol. 42 ı No. 6 ı 2007

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DF crystals show a similar emission spectrum (Figure 2A, orange curve) as the combined NBT/BCIP stain, peaking at 855 nm. This similarity in the spectra suggests that the majority of the optical signal from the stained embryos originates from NBT-DF. Nevertheless, it is the combination of the two reaction products that stabilize the NBT/BCIP stain, allowing us to take advantage of the fluorescence signal of this stain for in situ hybridization protocols. To rule out the possibility that the optical signal we observed is due to scattering of light by the precipitate (no wavelength shift) or leakage of light in the emission band by the excitation filter, we tested the performance of our filter set. We replaced the sample with a mirror to send 99% of the excitation light directly through the emission filter, representing a worst-case test for leakage. The mirror spectrum (Figure 1A, red curve) is nearly identical to the unstained and matrix background spectra, except for the peak at 670 nm due to leakage of the filter set. Because the NBT/BCIP and NBT-DF spectrum is more intense and distinctly different than the light leakage spectrum, we conclude that the optical signal is not due to simple leakage of light by our filter set. Photostability of a fluorescent stain is an important practical consideration for any imaging tool. To assess the bleach rate of the NBT/BCIP reaction product, we monitored the fluorescence intensity of the specimen under continuous illumination for 4 h at maximum intensity. The fluorescence intensity faded slowly, with a half-life of 27 min (Figure 2B). After 4 h of illumination, the initial dark purple color of the heavily stained region had bleached to yellow (compare top and bottom insets in Figure 2B). In summary, we report the heretofore undescribed fluorescence of the NBTDF/BCI reaction product derived from NBT/BCIP chromogenic staining protocols. We have demonstrated that it can be employed as a NIR fluorescence label that permits cellular resolution in whole-mount in situ hybridization studies. Although both components of the reaction product exhibit fluorescence, NBT-DF is the major NIR fluorophore in the specimens. The red Vol. 42 ı No. 6 ı 2007

excitation and NIR emission are ideal for use in whole-mounted specimens, as longer wavelengths are less scattered by tissue and autofluorescence is much lower in this wavelength range in most specimens; furthermore, the emission is easy to separate spectrally from the autofluorescence of the matrix tissue and from most fluorescent labels. Thus, the NIR emission will facilitate multilabel NBT/BCIP staining protocols in combination with visible fluorophores. We show here that current NBT/BCIP in situ hybridization protocols coupled to optical sectioning techniques result in single cell resolution without the development of new protocols for fluorescent in situ hybridization methods. ACKNOWLEDGMENTS

L.A.T., M.D.M., L.A.B., and D.W.M. contributed equally to this work. M.D.M. thanks the National Science Foundation (NSF) Louis Stokes Oklahoma Alliance for Minority Participation, Bridges to the Doctorate Program for a graduate research fellowship. This work was supported by the NSF CAREER grant no. CHE0239803, the Center for Physics in Nanostructures, NSF MRSEC no. DMR-0080054, Oklahoma EPSCoR, and NIH grants no. R01 HL078694 and P01 HD037105. COMPETING INTERESTS STATEMENT

The authors declare no competing interests. REFERENCES 1. Larsson, L.I., T. Christensen, and H. Dalboge. 1988. Detection of proopiomelanocortinm RNA by in situ hybridization, using a biotinylated oligodeoxynucleotide probe and avidin-alkaline phosphatase histochemistry. Histochemistry 89:109-116. 2. O’Keefe, H.P., D.A. Melton, B. Ferreiro, and C. Kinter. 1991. In situ hybridation. Meth. Cell Biol. 36:443-463. 3. Farquharson, M., R. Harvie, and A.M. McNicol. 1990. Detection of messenger RNA using a digoxigenin end labelled oligodeoxynucleotide probe. J. Clin. Pathol. 43:424-428. 4. Tautz, D. and C. Pfeifle. 1989. A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila

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Received 5 March 2007; accepted 6 April 2007. Address correspondence to Le Trinh, Beckman Institute (139-74), California Institute of Technology, 1200 E. California Blvd., Pasadena, CA 91125, USA. e-mail: [email protected]; and David McCauley, Department of Zoology, University of Oklahoma, 730 Van Vleet Oval, Norman, OK 73019, USA. e-mail: dwmccauley@ou. edu To purchase reprints of this article, contact: [email protected]

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