An immunocytochemical method for localization of estrogen receptors in rat tissues using a dinitrophenyl (DNP)-labeled rat monoclonal primary antibody

J. M W Gee, R. I. Nicholson, B. Jasani, G. R. Newman, W. M. Amselgruber

Research output: Contribution to journalArticle

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Abstract

We have developed an immunocytochemical method to demonstrate estrogen receptor in hormone-sensitive tissues of the rat using a dinitrophenyl (DNP) hapten-labeled rat anti-human estrogen receptor monoclonal antibody (MAb), H222. Mouse IgM anti-DNP was used secondarily, followed by a DNP/peroxidase conjugate, diaminobenzidine/hydrogen peroxide chromogen, and silver intensification. This method was applied to tissues from intact female rats and showed that estrogen receptor was localized in the nuclei of the stromal and glandular components of the uterine endometrium. Reduced receptor staining was observed in the luminal epithelium, with minimal myometrial staining. Anterior pituitary glands showed heterogeneous immunostaining and ovaries expressed the receptor predominantly in the interstitial cells; fallopian tubes demonstrated substantial epithelial staining. Uteri from chemically castrated rats showed reduced estrogen receptor immunostaining in both stromal and luminal cells, whereas staining was enhanced in the glandular elements. Classical estrogen-unresponsive tissues (heart, lung, and spleen) were unstained. Antibody controls involved pre-blocking antibody recognition sites on the receptor with unlabeled antibodies to estrogen receptor (H222, H226, and D547), as well as use of an inappropriate DNP-labeled antibody to metallothionein. These controls illustrated the specific nature of the DNP-H222 binding.

Original languageEnglish
Pages (from-to)69-78
Number of pages10
JournalJournal of Histochemistry and Cytochemistry
Volume38
Issue number1
Publication statusPublished - 1990
Externally publishedYes

Fingerprint

Estrogen Receptors
Monoclonal Antibodies
Staining and Labeling
Antibodies
Anterior Pituitary Gland
Blocking Antibodies
Fallopian Tubes
Haptens
Metallothionein
Stromal Cells
Endometrium
Silver
Hydrogen Peroxide
Uterus
Ovary
Estrogens
Spleen
Epithelium
Hormones
Lung

Keywords

  • DNP hapten sandwich staining
  • estrogen receptor
  • immunocytochemistry
  • monoclonal antibody
  • rat
  • silver intensification

ASJC Scopus subject areas

  • Anatomy
  • Cell Biology

Cite this

An immunocytochemical method for localization of estrogen receptors in rat tissues using a dinitrophenyl (DNP)-labeled rat monoclonal primary antibody. / Gee, J. M W; Nicholson, R. I.; Jasani, B.; Newman, G. R.; Amselgruber, W. M.

In: Journal of Histochemistry and Cytochemistry, Vol. 38, No. 1, 1990, p. 69-78.

Research output: Contribution to journalArticle

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