Triple-labeling method combining immunocytochemistry and in situ hybridization histochemistry: demonstration of overlap between Fos- immunoreactive and galanin mRNA-expressing subpopulations of luteinizing hormone-releasing hormone neurons in female rats

We describe a sensitive technique combining dual-label immunocytochemistry (ICC) with isotopic in situ hybridization histochemistry (ISHH). We developed this technique to characterize the receptor and/or peptide content of pheno-typically identified neurons that express cell markers of neuronal acti...

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Published inThe journal of histochemistry and cytochemistry Vol. 43; no. 4; pp. 363 - 370
Main Authors Hrabovszky, E, Vrontakis, ME, Petersen, SL
Format Journal Article
LanguageEnglish
Published United States Histochemical Soc 01.04.1995
SAGE Publications
Subjects
Animals
Autoradiography
Brain - metabolism
Female
Galanin
Gonadotropin-Releasing Hormone - analysis
Immunohistochemistry - methods
In Situ Hybridization - methods
Neurons - metabolism
Peptides - analysis
Proto-Oncogene Proteins c-fos - analysis
Rats
Rats, Sprague-Dawley
RNA, Messenger - analysis
Sensitivity and Specificity
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Summary:We describe a sensitive technique combining dual-label immunocytochemistry (ICC) with isotopic in situ hybridization histochemistry (ISHH). We developed this technique to characterize the receptor and/or peptide content of pheno-typically identified neurons that express cell markers of neuronal activity (immediate early gene products) after physiological or pharmacological perturbation. Tissue was fixed by perfusion with 4% paraformaldehyde in PBS, sucrose-infiltrated, and cryosectioned. Sections were stored in cryoprotectant or immediately hybridized. After stringent hybridization wash procedures, Fos and luteinizing hormone-releasing hormone (LHRH) neurons were visualized sequentially using immunocytochemistry. Finally, galanin mRNA was detected autoradiographically. We applied the technique to study of subpopulations of LHRH-containing neurons. Results of this study indicate that a majority of the LHRH neurons activated during the luteinzing hormone (LH) surge (as indicated by presence of nuclear Fos staining) also express mRNA encoding galanin. However, there is not a complete overlap between the subpopulation of LHRH neurons that express Fos and that which expresses galanin mRNA.
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ISSN:0022-1554
1551-5044
DOI:10.1177/43.4.7534782