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

E. Hrabovszky, M. E. Vrontakis, S. L. Petersen

Research output: Contribution to journalArticle

23 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)363-370
Number of pages8
JournalJournal of Histochemistry and Cytochemistry
Volume43
Issue number4
DOIs
Publication statusPublished - Jan 1 1995

Keywords

  • Fos
  • Galanin
  • Immediate early genes
  • Immunocytochemistry
  • Immunohistochemistry
  • In situ hybridization histochemistry
  • Luteinizing hormone-releasing hormone
  • Triple-labeling
  • mRNA

ASJC Scopus subject areas

  • Anatomy
  • Histology

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