Journal
JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY
Volume 48, Issue 10, Pages 1369-1375Publisher
HISTOCHEMICAL SOC INC
DOI: 10.1177/002215540004801007
Keywords
fluorescent in situ hybridization; tyramide signal amplification; immunohistochemistry; double labeling
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Funding
- NINDS NIH HHS [NS35404, NS35107] Funding Source: Medline
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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 alpha 2 (GFR alpha 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 alpha 2 mRNA and virtually all GFR alpha 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 alpha 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.
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