ARTICLE |
Correspondence to: Kevin A. Roth, Washington U. School of Medicine, Dept. of Pathology, Div. of Neuropathology, 660 S. Euclid Avenue, Box 8118, St Louis, MO 63110. E-mail: kroth@pathology.wustl.edu
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Summary |
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To understand the biological relationships among various molecules, it is necessary to define the cellular expression patterns of multiple genes and gene products. Relatively simple methods for performing multi-label immunohistochemical detection are available. However, there is a paucity of techniques for dual immunohistochemical (IHC) and mRNA in situ hybridization (ISH) detection. The recent development of improved non-radioactive detection systems and simplified ISH protocols has prompted us to develop a tyramide signal amplification method for sequential multi-label fluorescent ISH and IHC detection in either frozen or paraffin-embedded tissue sections. We used this method to examine the relationship between glial cell line-derived neurotrophic factor receptor 2 (GFR
2) mRNA expression and IHC localization of its co-receptor Ret in the trigeminal ganglion of postnatal Day 0 mice. We found that approximately 70% of Ret-immunoreactive neurons possessed GFR
2 mRNA and virtually all GFR
2-expressing neurons contained Ret-immunoreactive protein. Finally, we used paraformaldehyde-fixed, paraffin-embedded sections and a monoclonal antibody against neuron-specific nuclear antigen (NeuN) to demonstrate the neuronal specificity of GFR
2 mRNA expression in adult mouse brain. This multi-labeling technique should be applicable to a wide variety of tissues, antibodies, and probes, providing a relatively rapid and simple means to compare mRNA and protein localization. (J Histochem Cytochem 48:13691375, 2000)
Key Words: fluorescent in situ hybridization, tyramide signal amplification, immunohistochemistry, double labeling
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Introduction |
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IN SITU HYBRIDIZATION (ISH) detection of mRNAs is an invaluable tool for research and diagnostics, dramatically advancing the study of cell- and tissue-specific expression of many genes (
Historically, ISH was developed using radiolabeled probes (
Although most authors have used TSA ISH to detect DNA sequences, TSA has also been used to localize mRNA expression in the nervous system. TSA has proved more sensitive than AP deposition of BCIP/NBT for detection of oxytocin mRNA in rat brain sections and for peptide hormone mRNA detection in paraffin-embedded human tissue sections (
In this article we describe a method for performing dual mRNA ISH and antigen IHC on either frozen or paraffin-embedded sections using fluorescent TSA techniques. We used this method to examine the relationship between GFR2 mRNA expression and Ret protein immunoreactivity in the developing mouse nervous system and GFR
2 mRNA expression and NeuN immunoreactivity in the adult mouse brain.
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Materials and Methods |
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Tissue Preparation
Young adult and timed-pregnant SwissWebster mice were purchased from Harlan Sprague Dawley (Indianapolis, IN). Postnatal Day 0 and adult mice were deeply anesthetized with methoxyflurane (Metofane; Pitman-Moore, Mundelein, IL), decapitated, and their brains placed in embedding medium (Tissue Tek OCT compound; Miles, Elkhart, IN) and frozen on dry ice in liquid chlorodifluoromethane (HistoFreeze; Fisher Scientific, Pittsburgh, PA). Alternatively, tissues were fixed for 24 hr in 4% paraformaldehyde at room temperature (RT) and paraffin-embedded.
Probes, Antibodies, and Reagents
GFR2 cRNA probe was generated from a plasmid containing GFR
2 nucleotides 10021417 of GenBank accession number
AF002701 (
2 mRNA (
2 co-receptor Ret in postnatal Day 0 trigeminal ganglia and mouse anti-neuronal nuclei (NeuN) antibodies (Chemicon; Temecula, CA) were used to identify neurons by IHC in the adult brain.
TSA reagents were obtained from NEN Life Science Products. Direct fluorescence deposition was performed using TSA Plus Direct-Green or TSA Plus DirectCyanine 3. Indirect fluorescence deposition was performed with TSA Plus DNP (HRP) followed by TSA Plus DirectCyanine 3.
Dual GFR2 ISH and Ret IHC Detection on Frozen Sections
Sixteen-µm-thick coronal sections of postnatal Day 0 mouse brain were cut, air-dried, and fixed in 4% paraformaldehyde in PBS (10 mM phosphate, pH 7.2) for 30 min at 25C. All steps were performed at RT unless otherwise indicated. Sections were washed three times for 5 min each in PBS and incubated for 30 min in 0.1% active DEPC (in PBS). Sections were again washed in PBS and endogenous peroxidase activity was inhibited by incubation in 0.3% H2O2 (in PBS) for 30 min. Sections were then washed in PBS and incubated in 5 x SSC (0.75 M NaCl, 0.75 M Na-citrate) for 15 min. Tissue sections were then prehybridized for 2 hr at 58C in prehybridization buffer (5 x SSC, 50% formamide, pH to 7.5 with HCl, 50 µg/ml salmon sperm DNA). Slides were then incubated with digoxigenin-labeled probe (100500 ng/ml of probe in hybridization buffer) for 1224 hr at 58C. Posthybridization washes were performed with 2 x SSC and 0.1 x SSC (each for 1 hr at 65C) and sections were then washed in PBS and incubated for 30 min in PBSBB (PBS containing 1.0% bovine serum albumin, 0.2% powdered skim milk, and 0.3% Triton X-100). Digoxigenin-labeled GFR2 probe was localized with HRP-conjugated sheep anti-digoxigenin antibodies (Roche Molecular; Indianapolis, IN) diluted 1:1000 in PBSBB and incubated for 1 hr followed by PBS washes and TSA Plus DirectCyanine 3 deposition according to the manufacturer's protocol (NEN Life Science Products). If only single ISH detection was performed, slides were then washed in PBS, nuclei labeled with Hoechst 33258 (0.2 µg/ml; Sigma, St Louis, MO), sections mounted in PBS:glycerol (1:1), and viewed with a Zeiss Axioskop microscope equipped with epifluorescence.
For dual GFR2 ISH and Ret IHC localization, residual HRP activity from the initial TSA reaction described above was destroyed by incubation in 0.3% H2O2 in PBS for 10 min. Sections were then washed in PBS and nonspecific antibody binding was inhibited by a 30-min incubation in PBSBB. Rabbit anti-Ret antibodies (diluted 1:50 in PBSBB) were incubated on the sections for 1224 hr at 4C (alternatively, for 1 hr at RT) followed by PBS washes and HRP-conjugated donkey anti-rabbit antiserum (Jackson Immunoresearch Laboratories; West Grove, PA) diluted 1:1000 in PBSBB for 1 hr. Sections were again washed in PBS and reacted with TSA Plus DirectGreen according to the manufacturer's protocol (NEN Life Science Products). Sections were then incubated with Hoechst 33258, mounted in PBS:glycerol, and coverslipped.
Dual GFR2 ISH and NeuN IHC Detection on Paraffin-embedded Sections
Four-µm-thick paraffin-embedded brain sections were deparaffinized in xylene and rehydrated in isopropanol and water. Heat-induced epitope retrieval was then performed for 20 min in 0.01 M citrate buffer, pH 6.0. After a 20-min cool-down period, sections were washed in PBS and treated twice for 15 minutes each in 0.1% active DEPC in PBS. The remaining steps of the ISH detection protocol were identical to those described above for frozen sections. Localization of NeuN immunoreactivity (mouse anti-NeuN, 1:2000) was also performed as described above for Ret immunodetection.
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Results |
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GFR2 ISH and Ret IHC
On the basis of our previous ISH studies utilizing radiolabeled GFR2 cRNA (
2 expression in the trigeminal ganglia of postnatal Day 0 mice. Strong GFR
2 mRNA signal was localized to trigeminal neurons with TSA Plus DirectCyanine 3 detection (Fig 1A). Only weak or no signal was observed when the GFR
2 cRNA probe was omitted or when irrelevant digoxigenin-labeled cRNA probes were used in the procedure (Fig 1B). Increased GFR
2 mRNA signal intensity and/or optimal probe dilution could be achieved by extending the TSA Plus DirectCyanine 3 reaction to 12 hr or by performing TSA Plus DNP (HRP) followed by TSA Plus DirectCyanine 3 (data not shown).
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The tissue preparation method used for mRNA ISH was compatible with localization of Ret protein immunoreactivity in the trigeminal ganglion (Fig 1C). No reactivity was observed when primary antiserum was omitted from the procedure (data not shown). To determine the interrelationship between GFR2 mRNA and Ret protein expression in the trigeminal ganglion, we performed sequential GFR
2 mRNA ISH and Ret IHC using TSA Plus DirectCyanine 3 and TSA Plus DirectGreen. Both GFR
2 mRNA (Fig 1D) and Ret immunoreactivity (Fig 1E) were observed in a subset of trigeminal ganglion cells. Dual exposure of labeled sections revealed that virtually all GFR
2 mRNA-positive cells (111 of 112 cells evaluated) possessed Ret immunoreactivity but that approximately 30% of Ret-immunoreactive cells (50 of 161 cells evaluated) lacked detectable GFR
2 mRNA (Fig 1F). In control experiments, replacement of the rabbit anti-Ret antiserum with normal rabbit serum after GFR
2 ISH resulted in no specific labeling or co-localization with the GFR
2 mRNA signal, demonstrating the specificity of the Ret IHC localization.
GFR2 ISH and NeuN IHC
Digoxigenin- or biotin-conjugated GFR2 cRNA probes were used to detect GFR
2 mRNA in paraformaldehyde-fixed, paraffin-embedded adult mouse brain sections. Consistent with our previous study utilizing 33P-labeled cRNA probes (
2 mRNA was readily detected in many cell populations throughout the mouse central nervous system (Fig 2A). Weak or no reactivity was observed when the GFR
2 cRNA was omitted or when irrelevant biotin or digoxigenin labeled cRNAs were substituted (Fig 2B). Particularly intense GFR
2 reactivity was observed in the neocortex and hypothalamus. GFR
2 mRNA signal appeared localized to neurons on the basis of anatomic distribution and the nuclear features of positive cells. However, to unequivocally identify GFR
2 mRNA positive cells as neurons, we performed dual GFR
2 ISH and NeuN IHC localization. NeuN immunoreactivity is present in the vast majority of neurons in the brain and is absent in non-neuronal cell populations (
2 mRNA (Fig 2D) and NeuN immunoreactivity (Fig 2E) revealed neuron-specific GFR
2 mRNA expression (Fig 2F).
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Discussion |
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In this article we describe a relatively rapid and simple method for dual fluorescent ISH and IHC detection using TSA Plus reagents and either digoxigenin- or biotin-labeled cRNA probes. This method proved successful for at least three reasons. First, because proteinase K is not required during the ISH procedure (
The dual fluorescent ISH and IHC detection protocol was used to compare the expression patterns of GFR2 mRNA and Ret immunoreactivity in the postnatal Day 0 mouse trigeminal ganglion and GFR
2 mRNA and NeuN immunoreactivity in adult mouse brain. GFR
2 and Ret form a receptor complex that preferentially binds neurturin (NRTN), a member of the glial cell line-derived neurotrophic factor (GDNF) family of ligands (GFLs) (
s, an analysis of their expression patterns and that of the co-receptor Ret may shed light on the in vivo relationships between these molecules and provide clues to their physiological function. Our analysis of the trigeminal ganglion indicated that virtually all GFR
2 mRNA-expressing neurons possessed Ret-immunoreactivity but that approximately 30% of the Ret immunoreactive neurons lacked GFR
2 mRNA. These findings are consistent with our previous studies demonstrating that GFR
2 and Ret form a functional receptor for GFLs, particularly for NRTN, and that other members of the GFR
family, GFR
1 and GFR
3, are also expressed in the trigeminal ganglion (
1 and/or GFR
3 are co-expressed with Ret in the trigeminal neurons lacking GFR
2. Additional multi-labeling studies are required to directly test this hypothesis. In the adult mouse brain, GFR
2 mRNA was found in many neuronal populations, as previously described (
2 expression was directly demonstrated by co-localization of GFR
2 mRNA and NeuN immunoreactivity in single cells.
In this work, we used either digoxigenin- or biotin-labeled cRNA probes to detect GFR2 mRNA. However, we have also used HRP-conjugated oligonucleotides and a similar protocol to detect nestin mRNA in the developing mouse nervous system (data not shown). Our observations and the published literature suggest both advantages and disadvantages of oligonucleotide cDNA probes vs cRNA probes for mRNA ISH. Oligonucleotides are relatively inexpensive and can be designed to hybridize with virtually any available mRNA sequence, obviating the need to generate or obtain plasmids containing the targeted gene sequence. Their small size, typically 2550 nucleotides, facilitates tissue penetration and decreases or eliminates the requirement for tissue disruption before probe hybridization. However, DNARNA hybrids are less stable than RNARNA hybrids, specificity of binding is less stringent, and overall sensitivity is poor, necessitating the use of mixtures or "cocktails" of oligonucleotide sequences to achieve adequate detection (
In total, we have described a rapid, sensitive method for dual fluorescent mRNA in situ hybridization and immunohistochemistry using TSA Plus detection reagents. Additional studies are required to determine the applicability of this method to other probes, cells, and tissues.
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Acknowledgments |
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Supported in part by NEN Life Science Products and by National Institutes of Health grants NS35107 and NS35404. AUZ received fellowship support from the McDonnell Center for Cellular and Molecular Neurobiology.
We thank Cecelia Latham for technical assistance, Angela Schmeckebier for secretarial support, and Dr Mark Bobrow (NEN Life Science Products) for expert advice and insights on tyramide signal amplification.
Received for publication May 24, 2000; accepted May 24, 2000.
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