Summary
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1.
We have described a general ribonucleotide probein situ hybridization methodology for localization of mRNA in frozen, unfixed tissue sections of brain.
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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.
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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.
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4.
Tissue quality has been optimized for high-resolution anatomical localization of mRNA species by nuclear track emulsion.
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5.
The protocol is amenable to rapid, batchwise processing of tissue samples.
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References
Aronsson, M., Fuxe, K., Dong, Y., Agnati, L. F., Okret, S., and Gustafsson, J.-Å. (1988). Localization of glucocorticoid receptor mRNA in the male rat brain byin situ hybridization.Proc. Natl. Acad. Sci. USA 859331–9335.
Arriza, J. L., Simerly, R. B., Swanson, L. W., and Evans, R. M. (1988). The neuronal mineralocorticoid receptor as a mediator of glucocorticoid response.Neuron 1887–900.
Brahic, M., and Haase, A. T. (1978). Detection of viral sequences of low reiteration frequency byin situ hybridization.Proc. Natl. Acad. Sci. USA 756125–6129.
Conn, P. M. (1989).Methods in Enzymology, Vol. 168. Hormone Action. Part K. Neuroendrocrine Peptides, Academic Press, New York.
Fuxe, K., Wikström, A.-C., Okret, S., Agnati, L. F., Härfstrand, A., Yu, Z.-Y., Granholm, L., Zoli, M., Vale, W., and Gustafsson, J.-Å. (1985). Mapping of glucocorticoid receptor immunoreactive neurons in the rat tel- and diencephalon using a monoclonal antibody against the rat liver glucocorticoid receptor.Endocrinology 1171803–1812.
Gendelman, H. E. (1986). Appendix; D. Pretreatments. InIn Situ Hybridization in Brain (G. R. Uhl, Ed.), Plenum, New York, pp. 268–269.
Hayashi, S., Gillam, I. C., Delaney, A. D., and Tener, G. M. (1978). Acetylation of chromosome squashes ofDrosophila melanogaster decreases the background in autoradiographs from hybridization with [125I]-labeled RNA.J. Histochem. Cytochem. 26677–679.
Herkenham, M. (1988). Influence of tissue treatment on receptor autoradiography. InMolecular Neuroanatomy (F. W. Van Leeuwen, R. M. Buijs, C. W. Pool, and O. Pach, Eds.), Elsevier Science, New York, pp. 111–120.
Herkenham, M., and Pert, C. B. (1982). Light microscopic localization of brain opiate receptors: A general autoradiographic method which preserves tissue quality.J. Neurosci. 21129–1149.
McEwen, B. S., De Kloet, E. R., and Rostene, W. (1986). Adrenal steroid receptors and action in the nervous system.Physiol. Rev. 661121–1188.
Miesfeld, R., Rusconi, S., Godowski, P. J., Maler, B. A., Okret, S., Wikström, A. C., Gustafsson, J.-Å., and Yamamoto, K. R. (1986). Genetic complementation of a glucocorticoid receptor deficiency by expression of cloned receptor cDNA.Cell 46389–399.
Paxinos, G., and Watson, C. (1986).The Rat Brain in Stereotaxic Coordinates, 2nd ed., Academic Press, New York.
Perbal, B. (1988).A Practical Guide to Molecular Cloning, 2nd ed., John Wiley & Sons, New York, p. 443.
Pintar, J. E., and Lugo, D. I. (1987). Localization of peptide hormone gene expression in adult and embryonic tissue. InIn Situ Hybridization. Applications to Neurobiology (K. L. Valentino, J. H. Eberwine, and J. D. Barchas, Eds.), Oxford University Press, New York, pp. 179–196.
Rothman, R. B., Schumacher, U. K., and Pert, C. B. (1983). Binding of radiolabeled opiates to slide mounted sections of molded minced rat brain: A novel method for conducting radioreceptor assays.Neuropeptides 7283–288.
Schachter, B. S. (1987). Studies of neuropeptide expression gene expression in brain and pituitary. InIn Situ Hybridization. Applications to Neurobiology (K. L. Valentino, J. H. Eberwine, and J. D. Barchas, Eds.), Oxford University Press, New York, pp. 111–125.
Sousa, R. J., Tannery, N. H., and Lafer, E. M. (1989).In situ hybridization mapping of glucocorticoid receptor messenger ribonucleic acid in rat brain.Mol. Endocrinol. 3481–494.
Valentino, K. L., Eberwine, J. H., and Barchas, J. D. (1987).In Situ Hybridization. Applications to Neurobiology, Oxford University Press, New York.
Van Eekelen, J. A. M., Jiang, W., De Kloet, E. R., and Bohn, M. C. (1988). Distribution of the mineralocorticoid and the glucocorticoid receptor mRNAs in the rat hippocampus.J. Neurosci. Res. 2188–94.
Yang, G., Matocha, M. F., and Rapoport, S. I. (1988). Localization of glucocorticoid receptor messenger ribonucleic acid in hippocampus of rat brain usingin situ hybridization.Mol. Endocrinol. 2682–685.
Young, W. S., III (1989).In situ hybridization histochemical detection of neuropeptide mRNA using DNA and RNA probes. InMethods in Enzymology, Vol. 168. Hormone Action. Part K. Neuroendocrine Peptides, Academic Press, New York, pp. 702–710.
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Whitfield, H.J., Brady, L.S., Smith, M.A. et al. Optimization of cRNA probein situ hybridization methodology for localization of glucocorticoid receptor mRNA in rat brain: A detailed protocol. Cell Mol Neurobiol 10, 145–157 (1990). https://doi.org/10.1007/BF00733641
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DOI: https://doi.org/10.1007/BF00733641