RAPID COMMUNICATION |
Correspondence to: Denis G. Baskin, Div. of Endocrinology/Metabolism, Mail Stop 151, VA Puget Sound Health Care System, 1660 So. Columbian Way, Seattle, WA 98108. E-mail: baskindg@u.washington.edu
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Summary |
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To increase the sensitivity of fluorescence in situ hybridization (FISH) for detection of low-abundance mRNAs, we performed FISH on cryostat sections of rat hypothalamus with biotin-labeled riboprobes to leptin receptor (ObRb) and amplified the signal by combining tyramide signal amplification (TSA) and Enzyme-Labeled Fluorescent alkaline phosphatase substrate (ELF) methods. First, TSA amplification was done with biotinylated tyramide. Second, streptavidinalkaline phosphatase was followed by the ELF substrate, producing a bright green fluorescent reaction product. FISH signal for ObRb was undetectable when TSA or ELF methods were used alone, but intense ELF FISH signal was visible in hypothalamic neurons when the ELF protocol was preceded by TSA. The TSAELF was combined with FISH for pro-opiomelanocortin (POMC) and neuropeptide Y (NPY) mRNAs by hybridizing brain sections in a cocktail containing digoxigenin-labeled riboprobes to NPY or POMC mRNA and biotin-labeled riboprobes to ObRb mRNA. Dioxigenin-labeled NPY or POMC mRNA hybrids were subsequently detected first with IgGCy3. Then biotin-labeled leptin receptor hybrids were detected with the TSAELF method. Combining the ELF and TSA amplification techniques enabled FISH detection of scarce leptin receptor mRNAs and permitted the identification of leptin receptor mRNA in cells that also express NPY and POMC gene products.
(J Histochem Cytochem 48:15931599, 2000)
Key Words: leptin, NPY, POMC, brain, arcuate nucleus, obesity
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Introduction |
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Fluorescence in situ hybridization (FISH) techniques have been extensively applied to the histochemical mapping of specific nucleic acid sequences on chromosomes (
To identify cells that are direct targets of leptin, we recently combined ISH for ObRb, using a 33P-ObRb riboprobe, with FISH for neuropeptide transcripts, using digoxigenin- and biotin-labeled riboprobes to show that ObRb mRNA is expressed in neurons that contain mRNA for neuropeptide Y (NPY) and the pro-opiomelanocortin (POMC) precursor protein (
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Materials and Methods |
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Tissue Processing
Brains were obtained from anesthetized male Wistar rats (250300 g; n = 12) according to a protocol previously approved by the Animal Research Committees of the Seattle VA Puget Sound Health Care System Medical Center and the University of Washington. Brains were frozen immediately on dry ice and were stored at -80C until they were sectioned at 14 µm with a cryostat and mounted on RNAse-free slides. Sections were prepared for FISH with 4% paraformaldehyde, acetylation with acetic anhydride, dehydration with ethanol, and delipidation with chloroform, using standard protocols as described previously (
Riboprobes
Procedures for preparation and labeling of the riboprobes used in this study are described elsewhere (
Hybridization
Standard hybridization procedures were used, as described previously (
Double FISH
For the double FISH protocol, the sections were hybridized simultaneously for ObRb mRNA and a neuropeptide mRNA. A digoxigdenin-labeled riboprobe for either NPY mRNA or POMC mRNA and a biotin-labeled riboprobe for ObRb mRNA were mixed in a cocktail for the hybridization. After the posthybridization wash in SSC, slides were equilibrated in TNT buffer containing 0.1 M Tris (ph 7.4) 0.15 M NaCl, and 0.05% Triton X-100. A primary mouse anti-digoxigenin monoclonal antibody IgG (Jackson ImmunoResearch; West Grove, PA) diluted 1:5000 in TNT buffer with 1% normal goat serum was applied to each slide for 3 hr at 37C, followed by three washes in TNT buffer for 5 min each at RT and then goat anti-mouse IgGCy3 (Jackson ImmunoResearch) diluted 1:200 in TNT buffer for 1 hr at 37C and three washes as before. Sample sections were checked microscopically at this stage to verify labeling. The slides were then treated with the TSAELF protocol as described above and dipped in 1 µg/ml aqueous Hoechst 33258 (bisbenzimide) (Sigma) fluorochrome for visualization of cell nuclei (
Microscopy and Imaging
Visualization of FISH was done with a Zeiss Axioplan fluorescence microscope using a x63 oil immersion planapochromat objective. The red Cy3 fluorescence was visualized with a conventional rhodamine filter set, whereas the green fluorescence of the ELFalkaline phosphatase substrate reaction product was observed with a 320390-nm excitation filter, a 400-nm dichroic longpass filter, and a 535-nm barrier filter (Chroma Technology; Brattleboro, VT). Hoechst 33258 fluorescence of cell nuclei was observed with a 365-nm excitation filter, a 400-nm dichroic longpass filter, and a 420-nm barrier filter (Chroma Technology).
Digital RGB pseudocolored images (10 bit) of the fluorescence preparations were acquired with a Hamamatsu C4880 fast-cooled CCD camera (Hamamatsu; Tokyo, Japan) and the MCID imaging system (Imaging Research; St Catharines, Ont, Canada) and were exported to Adobe Photoshop (Tucson, AZ) as 300 dpi tiff RGB files. The images were processed with pseudocolor palettes that closely matched the intensity and contrast present in the original preparations, but no selective contrast enhancement of specific areas or cells was applied to the images. Image composites were prepared and labeled using Adobe Pagemaker.
Controls
Controls for the specificity of the FISH signal included (a) brain sections from characterized serial sets that had been previously hybridized with isotopic probes and oligonucleotide probes for these transcripts, (b) omission of the labeled riboprobes, and (c) RNase pretreatment. To confirm that the FISH was due to the combination of TSA and ELF, sections were hybridized with the following combinations: (a) no biotin-labeled riboprobes; (b) biotin-labeled riboprobes only (TSA and ELF omitted); (c) biotin-labeled riboprobes followed by TSA only (ELF omitted); and (d) biotin-labeled riboprobes plus ELF only (TSA omitted). To verify the improved sensitivity of the TSA-ELF protocol, riboprobes to ObRb mRNA were also transcribed with digoxigenin-UTP and hybridized with the Cy3 detection procedure that was used for NPY and POMC mRNA FISH, as described above. Levamisole was used to inhibit endogenous alkaline phosphatase activity.
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Results |
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The FISH protocol with the TSAELF procedure produced a granular fluorescent precipitate that was concentrated over neuronal cell bodies (Fig 1A). In many cases the precipitate completely obliterated the cell body, whereas in others it was a sparse sprinkling of precipitate. Nevertheless, this pattern of ELF reaction product was distinctive, enabling easy differentiation of labeled cells from scattered nonspecific background precipitate. The TSAELF FISH signal was also very bright, such that it was very easy to recognize and count individual labeled neurons by ObRb FISH at low magnification (x5 objective) because of the very bright fluorescence produced by the reaction product of the ELF alkaline phosphatase substrate. No FISH signal was present when the ObRb riboprobe was omitted or when RNase was used (Fig 1B). The TSAELF FISH procedure identified ObRb mRNA in many neuronal cell bodies throughout the caudal brainstem (Fig 1C).
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The TSAELF FISH procedure produced bright green signals when combined with red Cy3 fluorescence for NPY and POMC mRNAs in double FISH protocols. In the arcuate nucleus, many neuronal cell bodies contained the fluorescent green ELF reaction product when hybridized with the biotinylated riboprobes to ObRb mRNA. Some of these labeled neurons also showed red Cy3 fluorescence for POMC (Fig 1D) or NPY (Fig 1G) mRNA, indicating that a subset of arcuate nucleus POMC and NPY neurons express ObRb gene products.
FISH signal for ObRb was not detectable above background levels in the arcuate nucleus when the hybridization procedure was performed with either the TSA or the ELF protocol alone (Fig 1E), regardless of efforts to increase the signal by manipulations of the hybridization conditions or tissue preparation. However, when the ELF FISH protocol was preceded by TSA amplification, a bright FISH signal for ObRb mRNA was seen in the arcuate nucleus and the brainstem (Fig 1F). The FISH appeared as a fine punctate precipitate that was concentrated over the neuronal cell bodies.
No FISH signal above background was detected for ObRb mRNA when digoxigenin-labeled ObRb riboprobes and Cy3 immunodetection were used (not shown), regardless of hybridization or tissue fixation condition, whereas the NPY and POMC digoxigenin riboprobes produced strong FISH signals. No FISH signals were observed when riboprobes were omitted or when RNase was used. Omission of levamisole had no effect on the ELF FISH signal.
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Discussion |
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A major trend in FISH histochemistry over the past few years has been an emphasis on development of methods to enhance the visualization of mRNA copies that may be present in relatively low abundance in cells, either by increasing the numbers of mRNA copies to be detected (i.e., in situ PCR) or by amplification of hybridization reporter systems (
Recently,
An important development in FISH signal amplification was the introduction of fluorogenic alkaline phosphatase substrates, which enzymatically increase the fluorescent signal at the site of hybridization (
Although the ELF-97 method has the potential to be a very sensitive technique, its use requires careful attention to procedural details to avoid artifacts. The method is particularly predisposed to the formation of spurious fluorescent crystals, which obscure FISH signal and cause nonspecific deposition of crystals over cells and background. We found that the nonspecific formation of these crystals can be avoided by tightly adhering to the length of the ELF-97 reaction time, using the reagents in the manner suggested by the manufacturer, and using the mounting medium supplied with the kit. Because alkaline phosphatase is retained in the tissue after the reaction is stopped, we have found that high-pH mounting media should be avoided to prevent the nonspecific buildup of the fluorescent crystals over time. It is also important to use an inhibitor of alkaline phosphatase activity, such as levamisole, in the ELF blocking and wash buffers to inhibit endogenous alkaline phosphatase activity, because the ELF technique can also be used for histochemical detection of alkaline phosphatase activity (
Although both the ELF and the TSA techniques are useful by themselves in amplifying FISH signals, we found that they were ineffective for detection of the relatively low-abundance mRNAs encoding ObRb in the brain. The main new finding of our study is that a FISH signal that was undetectable with either of these methods alone was dramatically intensified to visual levels by preceding the ELF protocol with TSA amplification. The principle of the method is that the TSA procedure enzymatically catalyzes the deposition of more biotin molecules at the site of the biotinylated riboprobemRNA hybrids. This signal is then further amplified by binding streptavidinalkaline phosphatase to the biotin. Thus, the TSA procedure amplifies the accumulation of streptavidin-alkaline phosphatase at the site of hybridization. The subsequent ELF-97 alkaline phosphatase substrate is then dephosphorylated by alkaline phosphatase to yield a fluorescent precipitate, visually amplifying the hybridization reporter signal. Use of the TSA step before the ELF reaction generates a greater amount of fluorescent reaction product than would otherwise occur in the absence of the TSA amplification. This combined TSAELF procedure has potential for FISH detection of scarce mRNAs in cells and especially for double FISH of different transcripts that are expressed in the same cells.
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Acknowledgments |
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Supported by the Merit Review and Career Scientist Programs of the Department of Veterans Affairs Medical Research Service and by NIH grant DK-17047 to the Diabetes Endocrinology Research Center, University of Washington.
We are grateful to Gregory Cox of Molecular Probes for helpful suggestions.
Received for publication June 7, 2000; accepted October 4, 2000.
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