Pasinetti G M, Morgan D G, Johnson S A, Lerner S P, Myers M A, Poirier J, Finch C E
Andrus Gerontology Center, University of Southern California, Los Angeles 90089-0191.
Pharmacol Res. 1989 May-Jun;21(3):299-311. doi: 10.1016/s1043-6618(89)80008-8.
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.
本文描述了一种通过原位杂交结合免疫细胞化学（ICC）对同一切片上的酪氨酸羟化酶（TH）mRNA进行检测的方法。原位杂交方案针对[35S]cRNA（互补RNA，即反义链）探针浓度和杂交时间进行了优化。通过多个标准来衡量杂交的特异性。讨论了在原位放射自显影定量中测量颗粒密度与每细胞颗粒数的优势。最后，通过与印迹杂交比较，使用利血平诱导的肾上腺TH mRNA增加来验证原位杂交技术的定量方面。与肾上腺相反，通过这两种技术测量，利血平均未增加黑质（s. nigra）神经元中的TH mRNA。
