Kiyama H, Emson P C
MRC Group, AFRC Institute of Animal Physiology and Genetics Research, Cambridge, U.K.
Neuroscience. 1990;38(1):223-44. doi: 10.1016/0306-4522(90)90388-k.
The cellular localization of preprosomatostatin mRNA in the rat brain and sensory ganglia has been examined in detail using a newly developed highly sensitive non-radioactive in situ hybridization histochemistry procedure. An alkaline phosphatase labelled anti-sense 30mer oligodeoxynucleotide probe was used for detection of somatostatin mRNA. This probe readily demonstrated somatostatin gene expression throughout the rat CNS with very high contrast and good cellular localization. As a result, we visualized numerous somatostatin mRNA-positive cells in many CNS areas which had previously not been shown to contain a mRNA signal. This method detected a number of somatostatin mRNA-positive cells, in the mitral cell layer of accessory olfactory bulb, the glomerular layer of the main olfactory bulb, the dorsal part of the lateral septum, superficial gray layer of superior colliculus, inferior colliculus, anterior ventral cochlear nucleus, granular layer and Purkinje cell layer of cerebellum, and substantia gelatinosa of medulla and spinal cord, all areas where signal detection using radiolabelled in situ probes has previously been rather difficult. The principle advantages of the present method include the very precise cellular resolution of signal, the rapid reaction time and low background. The sensitivity of the present method seems to be at least equivalent to most immunocytochemical procedures and more sensitive than most isotopic in situ hybridization methods.
利用新开发的高灵敏度非放射性原位杂交组织化学方法,已对大鼠脑和感觉神经节中前促生长抑素原mRNA的细胞定位进行了详细研究。使用碱性磷酸酶标记的反义30聚体寡脱氧核苷酸探针检测生长抑素mRNA。该探针能很容易地在整个大鼠中枢神经系统中显示出生长抑素基因表达,对比度非常高,细胞定位良好。结果,我们在许多以前未显示含有mRNA信号的中枢神经系统区域中观察到大量生长抑素mRNA阳性细胞。该方法在副嗅球的二尖瓣细胞层、主嗅球的肾小球层、外侧隔的背侧部分、上丘的浅灰质层、下丘、前腹侧蜗神经核、小脑的颗粒层和浦肯野细胞层以及延髓和脊髓的胶状质中检测到许多生长抑素mRNA阳性细胞,而使用放射性标记原位探针以前在所有这些区域进行信号检测都相当困难。本方法的主要优点包括信号的细胞分辨率非常精确、反应时间快和背景低。本方法的灵敏度似乎至少与大多数免疫细胞化学方法相当,且比大多数同位素原位杂交方法更灵敏。