Article

Cellular and Molecular Neurobiology

, Volume 10, Issue 1, pp 145-157

Optimization of cRNA probein situ hybridization methodology for localization of glucocorticoid receptor mRNA in rat brain: A detailed protocol

  • Harvey J. WhitfieldJr.Affiliated withUnit on Functional Neuroanatomy, Clinical Neuroendocrinology Branch, NIMH
  • , Linda S. BradyAffiliated withUnit on Functional Neuroanatomy, Clinical Neuroendocrinology Branch, NIMH
  • , Mark A. SmithAffiliated withUnit on Functional Neuroanatomy, Clinical Neuroendocrinology Branch, NIMH
  • , Evagelia MamalakiAffiliated withUnit on Functional Neuroanatomy, Clinical Neuroendocrinology Branch, NIMH
  • , Robert J. FoxAffiliated withUnit on Functional Neuroanatomy, Clinical Neuroendocrinology Branch, NIMH
  • , Miles HerkenhamAffiliated withUnit on Functional Neuroanatomy, Clinical Neuroendocrinology Branch, NIMH

Rent the article at a discount

Rent now

* Final gross prices may vary according to local VAT.

Get Access

Summary

  1. 1.

    We have described a general ribonucleotide probein situ hybridization methodology for localization of mRNA in frozen, unfixed tissue sections of brain.

     
  2. 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. 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. 4.

    Tissue quality has been optimized for high-resolution anatomical localization of mRNA species by nuclear track emulsion.

     
  5. 5.

    The protocol is amenable to rapid, batchwise processing of tissue samples.

     

Key words

complementary RNA (cRNA) probe densitometry glucocorticoid receptor (rGR) mRNA hippocampus locus ceruleus mineralocorticoid receptor (rMR) mRNA ribonucleotide probein situ hybridization