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Fluorescence In Situ Hybridization of Scarce Leptin Receptor mRNA using the Enzyme-Labeled Fluorescent Substrate Method and Tyramide Signal Amplification

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

	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, streptavidin–alkaline 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 TSA–ELF 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 IgG–Cy3. Then biotin-labeled leptin receptor hybrids were detected with the TSA–ELF 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 48:1593–1599, 2000)

Key Words: leptin, NPY, POMC, brain, arcuate nucleus, obesity

	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 (Speel et al. 1999 ), but relatively few studies have used FISH for detection of mRNA. An advantage of FISH, however, is the potential for simultaneous detection of multiple mRNA species in a single tissue section or cell, as has been shown for neuropeptide mRNAs in the hypothalamus (Trembleau and Bloom 1995 ; Hahn et al. 1998 ; Baskin et al. 1999a ) and gastrointestinal system (Larsson and Hougaard 1991 ). Although FISH techniques have been used successfully to visualize the presence of mRNA for receptors and transporters that are relatively highly expressed (Williams and Beitz 1993 ; Sibon et al. 1996 ; Gao et al. 1999 ), we have found that conventional FISH procedures are insufficiently sensitive to detect mRNAs that are present in relatively low abundance, such as those that encode leptin receptors. Leptin is the product of the Ob gene in adipose tissue cells. Its major function is to signal the status of caloric intake and adipose tissue mass to the brain, thus reducing food intake and body weight (Baskin et al. 1999b ; Schwartz et al. 2000 ). One potential marker for brain cells targeted by leptin is the long-form splice variant of the leptin receptor (ObRb), which has been demonstrated in neuronal cell bodies of the hypothalamus by radioactive ISH (Mercer et al. 1996 ; Elmquist et al. 1998 ; Baskin et al. 1998 , Baskin et al. 1999a ) and immunocytochemistry (Baskin et al. 1999c ).

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 (Schwartz et al. 1996 , Schwartz et al. 1997 ). Because the relatively low cellular resolution of isotopic ISH was not favorable for precise cellular localization of ObRb mRNA, we attempted to use FISH to localize these transcripts. However, we found that the conventional FISH protocols we had successfully used for detection of neuropeptide mRNAs in the brain (Hahn et al. 1998 ; Baskin et al. 1998 , Baskin et al. 1999a , Baskin et al. 1999c ), including the tyramide signal amplification (TSA) technique (Adams 1992 ), did not reveal ObRb mRNA. Therefore, we adopted the Enzyme-Labeled Fluorescent (ELF) method, which employs a highly fluorescent alkaline phosphatase substrate (Singer et al. 1994 ) and has been used for amplification of immunocytochemical and ISH signals (Larison et al. 1995 ; Paragas et al. 1997 ). We found that combining the ELF and TSA methods resulted in a very bright FISH signal for ObRb mRNA, whereas use of either the ELF or the TSA methods alone was ineffective. Although this report describes the protocol and its use specifically for ObRb mRNA FISH, the method appears to have general applicability for FISH detection of mRNAs that are present in low abundance. We recently used this TSA–ELF FISH method to identify NPY and POMC cells that express scarce mRNAs encoding ObRb and SOCS-3 proteins in the hypothalamus (Baskin et al. 2000 ).

	Materials and Methods

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Tissue Processing
Brains were obtained from anesthetized male Wistar rats (250–300 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 (Schwartz et al. 1996 ; Baskin et al. 1998 , Baskin et al. 1999a ).

Riboprobes
Procedures for preparation and labeling of the riboprobes used in this study are described elsewhere (Schwartz et al. 1997 , Schwartz et al. 1998 ; Baskin et al. 1998 , Baskin et al. 1999c , Baskin et al. 2000 ). The ObRb mRNA riboprobes were prepared with biotin-UTP by transcribing cDNA containing the specific coding region for the cytoplasmic tail of the ObRb splice variant (Baskin et al. 1998 , Baskin et al. 1999a ). Riboprobes used to detect NPY and POMC mRNAs were transcribed with digoxigenin-UTP (Hahn et al. 1998 ; Baskin et al. 1999a , Baskin et al. 2000 ).

Hybridization
Standard hybridization procedures were used, as described previously (Schwartz et al. 1997 , Schwartz et al. 1998 ; Baskin et al. 1998 , Baskin et al. 1999a , Baskin et al. 1999c , Baskin et al. 2000 ). For brevity, only the essential details for performing the FISH TSA–ELF protocol are described here. FISH with biotin-labeled riboprobes complementary to mRNA encoding ObRb was visualized in a two-step process. In the first step, the biotin moiety was amplified using a Renaissance TSA-Indirect Kit (NEN Life Sciences; Boston, MA) according to the manufacturer's protocol. Briefly, streptavidin–HRP was diluted 1:100 with TNT buffer and applied to the slides, which were incubated for 30 min at room temperature (RT) in a moist chamber. Slides were then washed three times for 5 minutes each in TNT buffer at RT, followed by application of biotinyl tyramide diluted 1:50 in the amplification buffer provided with the kit. After incubation for 10 min at RT in a moist chamber, the slides were washed three times in TNT buffer for 5 min each at RT. In the second step of the procedure, the biotin deposited as a result of the TSA amplification was further amplified and visualized by ELF, using and ELF-97 mRNA In Situ Hybridization Kit #2 (Molecular Probes; Eugene, OR). Slides were placed in blocking buffer containing 30 mM Tris (ph 7.4), 150 mM NaCl, 1% BSA, 0.5% Triton X-100, and 1 mM levamisole (Sigma; St Louis, MO) for 30 min at RT in a moist chamber. Streptavidin–alkaline phosphatase conjugate (supplied with the ELF kit) was diluted 1:50 in TNT buffer and applied to the slides for 30 min at RT. Slides were then washed three times for 5 min each at RT in a pre-reaction buffer containing 30 mM Tris (ph 7.4) with 150 mM NaCl. This buffer removes residual BSA and detergent, both of which inhibit the ELF reaction. The ELF substrate was diluted 1:20 with the buffer supplied with the kit, then filtered with a 0.2-µm spin filter. Additives A and B in the kit were added to the filtrate at a dilution of 1:500. The final substrate solution was applied to each slide for exactly 10 min at RT in a moist chamber. The slides were then immediately rinsed with a stop buffer containing 100 mM Tris (ph 7.4), 25 mM EDTA, 0.5% Triton X-100, and 1 mM levamisole, followed by washing three times for 5 min each at RT in stop buffer. The slides were blotted to remove excess liquid, then coverslipped using the mounting medium supplied with the kit.

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 IgG–Cy3 (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 TSA–ELF protocol as described above and dipped in 1 µg/ml aqueous Hoechst 33258 (bisbenzimide) (Sigma) fluorochrome for visualization of cell nuclei (Araki et al. 1987 ).

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 ELF–alkaline phosphatase substrate reaction product was observed with a 320–390-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.

	Results

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The FISH protocol with the TSA–ELF 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 TSA–ELF 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 TSA–ELF FISH procedure identified ObRb mRNA in many neuronal cell bodies throughout the caudal brainstem (Fig 1C).

View larger version (83K): [in this window] [in a new window]   Figure 1. TSA–ELF FISH for ObRb mRNA in neuron cell bodies. (A) Hypoglossal nucleus of the rat caudal brainstem. The TSA–ELF procedure produced bright fluorescent precipitate over neuronal cell bodies (one indicated by arrow), seen in transverse section of the brainstem. TSA–ELF FISH signal was digitally captured by the MCID imaging program and is shown in pseudocolor green. Background is not detectable. (B) RNase control (section adjacent to A) showing low, almost undetectable, background and nonspecific signal. Position of fourth ventricle is indicated (V4). Bar = 100 µm. (C) TSA–ELF FISH for ObRb mRNA in neuron cell bodies of the trigeminal nucleus of the brainstem. The ELF reaction product appears as a bright yellow-green fluorescent signal in the cell bodies of labeled neurons. Nuclei show blue fluorescence of the Hoechst 33258 fluorochrome. The image was captured photographically as a transparency and scanned into digital format. (D) TSA–ELF FISH co-localization of mRNAs for ObRb (green ELF fluorescence) and pro-opiomelanocortin protein (POMC) (red Cy3 fluorescence) in the hypothalamic arcuate nucleus. Some POMC neurons show patches of ObRb FISH (yellow) where green and red fluorescence pseudocolors coincide. Cells with FISH signal for ObRb mRNA but not for POMC mRNA may express other peptides such as NPY. (E–G) Combining the TSA–ELF protocol for ObRb mRNA with FISH for NPY mRNA. No ObRb mRNA FISH signal was detected when ELF (E) or TSA (not shown) was used alone. Combining the TSA and ELF procedures produced a bright green fluorescent precipitate (shown in pseudocolor) over neuronal cell bodies in the arcuate nucleus (F) and permitted co-localization with NYP mRNA, as shown by red cy3 fluorescence (G). Neurons that co-expressed ObRb and NPY mRNAs are indicated by yellow pseudocolor in G. Bars = 50 µm.

The TSA–ELF 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.

	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 (Speel et al. 1999 ) Of these, signal amplification methods have become more widely adopted for demonstrating low-abundance transcripts by FISH, and several strategies have been employed for this purpose. Approaches that have been successfully used include mixtures of several fluorescent-labeled oligonucleotides directed at different coding regions of a transcript (Trembleau and Bloom 1995 ), riboprobes labeled with digoxigenin or biotin and revealed with Cy3–IgG or streptavidin–Cy3 (Hahn et al. 1998 ; Baskin et al. 1999c , Baskin et al. 2000 ) or fluorescein (Durrant et al. 1995 ), BrdU-labeled DNA probes (Kitazawa et al. 1989 ), rhodamine-labeled latex microspheres (Senatorov et al. 1997 ), horseradish peroxidase-labeled oligodeoxynucleotides combined with TSA (van de Corput et al. 1998 ), and catalyzed reporter deposition (CARD) (a synonym for TSA) with tyramine derivatives (Speel et al. 1999 ). A rapid and sensitive method for combining FISH and fluorescence immunocytochemistry using TSA Plus reagents (NEN Life Sciences; Boston, MA) was recently reported by Zaidi et al. 2000 .

Recently, Yang et al. 1999 reported that the combination of TSA and alkaline phosphatase for FISH with digoxigenin-labeled riboprobes yielded strong hybridization signals for mRNAs that are expressed at very low abundance, thereby achieving the high sensitivity normally associated with radioactive probes but with the cellular resolution provided by chromogenic enzymatic detection. The TSA–ELF method described here is a conceptual extension of many of the foregoing reports, particularly of the use of alkaline phosphatase as a reporter system. The novel aspect of our method is the use of a fluorogenic alkaline phosphatase substrate instead of a chromogenic substrate, thereby significantly enhancing the sensitivity of FISH for detection of mRNA present in relatively low abundance.

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 (Larsson and Hougaard 1991 ; Speel et al. 1992 ). In the present study, we elected to use the ELF–alkaline phosphatase method because of our previous success with ELF fluorescence in double immunocytochemical staining (Baskin et al. 1995 ). The ELF-97 alkaline phosphatase substrate (2-(5'-chloro-2-phosphoryloxyphenyl)-6-chloro-4(3H)-quinazolinone), has characteristics that make it favorable for this purpose (Singer et al. 1994 ; Paragas et al. 1997 ). First, when used with appropriate filters, it emits a very bright green fluorescent signal that is comparable to that produced by Cy3 but has excitation and emission maxima that are widely separated from those of Cy3, with no "bleed-through" of signals. Therefore, it can be used as a green fluorochrome to complement the red fluorescence of Cy3 in double labeling protocols (Baskin et al. 1995 ).

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 (Cox and Singer 1999 ; Telford et al. 1999 ). The timing of the ELF-97 reaction also appears to be critical, because the rate of the reaction is rapid, sometimes on the order of seconds, and allowing it to continue too long can result in formation of large amounts of spurious background crystals (Larison et al. 1995 ; Paragas et al. 1997 ). These can migrate away from the original site of deposition and cause spontaneous crystal growth, further increasing the nonspecific background signal. The manufacturer warns against exposing the precipitate to organic solvents, which cause it to dissolve. We have observed that the reaction product also dissolves in the presence of excess TNT buffer.

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 riboprobe–mRNA hybrids. This signal is then further amplified by binding streptavidin–alkaline 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 TSA–ELF 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.

	Acknowledgments

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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