Elsevier

Pharmacological Research

Volume 21, Issue 3, May–June 1989, Pages 299-311
Pharmacological Research

Combined in situ hybridization and immunocytochemistry in the assay of pharmacological effects on tyrosine hydroxylase mRNA concentration

https://doi.org/10.1016/S1043-6618(89)80008-8Get rights and content

Summary

An assay for tyrosine hydroxylase (TH) mRNA by in situ hybridization in combination with immunocytochemistry (ICC) for TH on the same section is described. The in situ hybridization protocol was optimized for [35S]cRNA (complementary RNA, i.e. anti-sense strand) probe concentration and time of hybridization. The specificity of hybridization was measured by several critera. The advantage of measuring grain density versus grains per cell is discussed for quantitation of in situ autoradiography. Finally, the reserpine-induced increase in adrenal TH mRNA was used to validate quantitative aspects of the in situ hybridization technique by comparison with blot hybridization. In contrast to the adrenal, reserpine did not increase TH mRNA in substantia nigra (s. nigra) neurons as measured by either technique.

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    Control brain slides were hybridized, exposed and developed simultaneously with the experimental slides. Uniformly these control slides demonstrated the known patterns of gene expression in striatopallidal neurons for A2a-adenosine and D2-dopamine receptor [39,15], cortical neurons for A1-adenosine receptors [40], and nigrostriatal neurons for TH [31] (data not shown). In addition, the patterns of TH and D2-dopamine, A2a-adenosine and A1-adenosine receptor mRNA expression differed in the tissue bloc of the carotid body and petrosal ganglion, further demonstrating probe-specificity.

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