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

Abstract

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 phenotypically 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 luteinizing 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.