Optimization of cRNA probein situ hybridization methodology for localization of glucocorticoid receptor mRNA in rat brain: A detailed protocol |
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Authors: | Harvey J. Whitfield Jr. Linda S. Brady Mark A. Smith Evagelia Mamalaki Robert J. Fox Miles Herkenham |
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Affiliation: | 1. Unit on Functional Neuroanatomy, Clinical Neuroendocrinology Branch, NIMH, Building 36, Room 2D15, 20892, Bethesda, Maryland, USA
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Abstract: | 1. | We have described a general ribonucleotide probein 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. |
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Keywords: | complementary RNA (cRNA) probe densitometry glucocorticoid receptor (rGR) mRNA hippocampus locus ceruleus mineralocorticoid receptor (rMR) mRNA ribonucleotide probein situ hybridization |
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