Optimization of cRNA probe in situ hybridization methodology for localization of glucocorticoid receptor mRNA in rat brain: a detailed protocol.
1. We have described a general ribonucleotide probe in situ hybridization methodology for localization of mRNA in frozen, unfixed tissue sections of brain. 2. The most important steps in obtaining consistent and reproducible autoradiographs with ribonucleotide probes were tissue acetylation and application of the radiolabeled probe to tissue sections under unsealed, glass coverslips. 3. Variability of the hybridization signal in tissue sections has been minimized to achieve a high degree of reproducibility within a given experiment as determined by densitometric analysis of rat glucocorticoid and mineralocorticoid receptor mRNA hybridization autoradiographs. 4. Tissue quality has been optimized for high-resolution anatomical localization of mRNA species by nuclear track emulsion. 5. The protocol is amenable to rapid, batchwise processing of tissue samples.
Whitfield
H J
HJ
Clinical Neuroendocrinology Branch, NIMH, Bethesda, Maryland 20892.
Brady
L S
LS
Smith
M A
MA
Mamalaki
E
E
Fox
R J
RJ
Herkenham
M
M
D000818:Animals; D001921:Brain / Q000378:metabolism*; D008297:Male; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D011965:Receptors, Glucocorticoid / Q000235:genetics* / Q000378:metabolism
D009841:Oligonucleotides; D012333:RNA, Messenger; D011965:Receptors, Glucocorticoid