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

• Published in: The journal of histochemistry and cytochemistry Vol. 43; no. 4; pp. 363 - 370
• Main Authors: Hrabovszky, E, Vrontakis, ME, Petersen, SL
• Format: Journal Article
• Language: English
• 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
• ISSN: 0022-1554; 1551-5044
• DOI: 10.1177/43.4.7534782

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.