Microwave Strategy for Improving the Simultaneous Detection of Estrogen Receptor and Galanin Receptor mRNA in the Rat Hypothalamus

In a attempt to improve the sensitivity of the simultaneous use of immunohistochemistry (IHC) with estrogen receptor (ER) and in situ hybridization (ISH) with a neuropeptide receptor, we first applied an existing microwave (MW) irradiation protocol for immunohistochemical detection of the estrogen r...

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Bibliographic Details
Published in:The journal of histochemistry and cytochemistry 2001-07, Vol.49 (7), p.901-910
Main Authors: Mitchell, Valerie, Feyereisen, Karine, Bouret, Sebastien, Leroy, Daniele, Beauvillain, Jean-Claude
Format: Article
Language:English
Subjects:
Animals
Female
Frozen Sections
Hypothalamus - anatomy & histology
Hypothalamus - metabolism
Immunohistochemistry - methods
In Situ Hybridization - methods
Microwaves
Rats
Rats, Wistar
Receptors, Estrogen - metabolism
Receptors, Galanin
Receptors, Neuropeptide - metabolism
RNA, Messenger - metabolism
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Summary:In a attempt to improve the sensitivity of the simultaneous use of immunohistochemistry (IHC) with estrogen receptor (ER) and in situ hybridization (ISH) with a neuropeptide receptor, we first applied an existing microwave (MW) irradiation protocol for immunohistochemical detection of the estrogen receptor in frozen brain sections. Regions of interest were the preoptic area and the arcuate nucleus of the hypothalamus. ER signal was effective only after MW heating of sections in the two regions. Control sections without pretreatment exhibited no staining for ER. Second, the MW protocol was applied in a novel procedure that consists of evaluation of the expression of the galanin receptor mRNA with a radioactive riboprobe after MW pretreatment. The galanin receptor mRNA signal intensity obtained after heating was quantitatively at least as good or significantly increased according to the region, with no discernible loss of tissue morphology. Finally, we describe a novel application of MW pretreatment on the same frozen section processed with ER antibody and a radioactive galanin receptor riboprobe. The stainings for estrogen and galanin receptors were intense in many cells of the preoptic area, with very low background. These results show that both IHC and ISH can be significantly improved by subjecting frozen sections to MW heating before the double labeling. This approach may provide a potential method to answer the important question of whether or not estrogen has a direct action on the expression of a peptide receptor.
ISSN:0022-1554
1551-5044
DOI:10.1177/002215540104900710