Optimization of cRNA probe in situ hybridization methodology for localization of glucocorticoid receptor mRNA in rat brain: a detailed protocol.

Cellular and molecular neurobiology ·Vol. 10 ·No. 1 ·1990-03-00 ·Pages 145-57

Whitfield HJ, Brady LS, Smith MA, Mamalaki E, Fox RJ, Herkenham M

Abstract

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.

MeSH Terms

Animals Brain/metabolism Male Nucleic Acid Hybridization Oligonucleotides RNA, Messenger/genetics,metabolism Rats Rats, Inbred Strains Receptors, Glucocorticoid/genetics,metabolism

Chemicals

Oligonucleotides RNA, Messenger Receptors, Glucocorticoid

Authors & Affiliations

6 authors, click to expand affiliations / ORCID

Whitfield H J

Clinical Neuroendocrinology Branch, NIMH, Bethesda, Maryland 20892.

Brady L S

Smith M A

Mamalaki E

Fox R J

Herkenham M

References (13)

13 references, click to expand

Genetic complementation of a glucocorticoid receptor deficiency by expression of cloned receptor cDNA.
Cell. 1986 Aug 1;46(3):389-99 PMID: 3755378
Adrenal steroid receptors and actions in the nervous system.
Physiol Rev. 1986 Oct;66(4):1121-88 PMID: 3532143
Localization of glucocorticoid receptor messenger ribonucleic acid in hippocampus of rat brain using in situ hybridization.
Mol Endocrinol. 1988 Aug;2(8):682-5 PMID: 3211154
Localization of glucocorticoid receptor mRNA in the male rat brain by in situ hybridization.
Proc Natl Acad Sci U S A. 1988 Dec;85(23):9331-5 PMID: 3194428
Detection of viral sequences of low reiteration frequency by in situ hybridization.
Proc Natl Acad Sci U S A. 1978 Dec;75(12):6125-9 PMID: 216014
Acetylation of chromosome squashes of Drosophila melanogaster decreases the background in autoradiographs from hybridization with [125I]-labeled RNA.
J Histochem Cytochem. 1978 Aug;26(8):677-9 PMID: 99471
Binding of radiolabeled opiates to slide-mounted sections of molded minced rat brain: a novel method for conducting radioreceptor assays.
Neuropeptides. 1983 Oct;3(6):493-9 PMID: 6320041
In situ hybridization histochemical detection of neuropeptide mRNA using DNA and RNA probes.
Methods Enzymol. 1989;168:702-10 PMID: 2725319
In situ hybridization mapping of glucocorticoid receptor messenger ribonucleic acid in rat brain.
Mol Endocrinol. 1989 Mar;3(3):481-94 PMID: 2747654
Distribution of the mineralocorticoid and the glucocorticoid receptor mRNAs in the rat hippocampus.
J Neurosci Res. 1988 Sep;21(1):88-94 PMID: 2851057
Light microscopic localization of brain opiate receptors: a general autoradiographic method which preserves tissue quality.
J Neurosci. 1982 Aug;2(8):1129-49 PMID: 6286904
Mapping of glucocorticoid receptor immunoreactive neurons in the rat tel- and diencephalon using a monoclonal antibody against rat liver glucocorticoid receptor.
Endocrinology. 1985 Nov;117(5):1803-12 PMID: 4042964
The neuronal mineralocorticoid receptor as a mediator of glucocorticoid response.
Neuron. 1988 Nov;1(9):887-900 PMID: 2856104

Article Info

Journal

Cellular and molecular neurobiology

Abbr.

Cell Mol Neurobiol

ISSN

0272-4340

DOI

10.1007/BF00733641

Published

1990-03-00

Pages

145-57

Language

English

Region

United States

NLM ID

8200709

Subset

External Links

PubMed source DOI: 10.1007/BF00733641 View journal details

Analysis Services

Optimization of cRNA probe in situ hybridization methodology for localization of glucocorticoid receptor mRNA in rat brain: a detailed protocol.

Contact

WeChat Official Account