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 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. (J Histochem Cytochem 49:901-910, 2001)
Mitchell
V
V
INSERM U422, Institut Fédératif de Recherches 22, Laboratoire de Neuroendocrinologie et Physiopathologie Neuronale, Lille, France. mitchell@biserte.lille.inserm.fr
Feyereisen
K
K
Bouret
S
S
Leroy
D
D
Beauvillain
J C
JC
D000818:Animals; D005260:Female; D005629:Frozen Sections; D007031:Hypothalamus / Q000033:anatomy & histology / Q000378:metabolism*; D007150:Immunohistochemistry / Q000379:methods; D017403:In Situ Hybridization / Q000379:methods; D008872:Microwaves; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D011960:Receptors, Estrogen / Q000378:metabolism*; D044088:Receptors, Galanin; D018013:Receptors, Neuropeptide / Q000378:metabolism*
D012333:RNA, Messenger; D011960:Receptors, Estrogen; D044088:Receptors, Galanin; D018013:Receptors, Neuropeptide