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

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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
Publication statusPublished - 1995

Fingerprint

Galanin
Gonadotropin-Releasing Hormone
In Situ Hybridization
Immunohistochemistry
Neurons
Messenger RNA
Immediate-Early Genes
Peptide Receptors
Luteinizing Hormone
Sucrose
Perfusion
Pharmacology
Staining and Labeling

Keywords

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

ASJC Scopus subject areas

  • Cell Biology
  • Anatomy

Cite this

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title = "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.",
keywords = "Fos, Galanin, Immediate early genes, Immunocytochemistry, Immunohistochemistry, In situ hybridization histochemistry, Luteinizing hormone-releasing hormone, mRNA, Triple-labeling",
author = "E. Hrabovszky and Vrontakis, {M. E.} and Petersen, {S. L.}",
year = "1995",
language = "English",
volume = "43",
pages = "363--370",
journal = "Journal of Histochemistry and Cytochemistry",
issn = "0022-1554",
publisher = "Histochemical Society Inc.",
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T1 - Triple-labeling method combining immunocytochemistry and in situ hybridization histochemistry

T2 - Demonstration of overlap between Fos- immunoreactive and galanin mRNA-expressing subpopulations of luteinizing hormone-releasing hormone neurons in female rats

AU - Hrabovszky, E.

AU - Vrontakis, M. E.

AU - Petersen, S. L.

PY - 1995

Y1 - 1995

N2 - 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.

AB - 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.

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KW - Immediate early genes

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KW - Immunohistochemistry

KW - In situ hybridization histochemistry

KW - Luteinizing hormone-releasing hormone

KW - mRNA

KW - Triple-labeling

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