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 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.
Hrabovszky
E
E
Department of Biology, University of Massachusetts, Amherst.
Vrontakis
M E
ME
Petersen
S L
SL
D000818:Animals; D001345:Autoradiography; D001921:Brain / Q000378:metabolism; D005260:Female; D019004:Galanin; D007987:Gonadotropin-Releasing Hormone / Q000032:analysis*; D007150:Immunohistochemistry / Q000379:methods*; D017403:In Situ Hybridization / Q000379:methods*; D009474:Neurons / Q000378:metabolism*; D010455:Peptides / Q000032:analysis*; D016760:Proto-Oncogene Proteins c-fos / Q000032:analysis*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity
D010455:Peptides; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D007987:Gonadotropin-Releasing Hormone; D019004:Galanin