Analysis of GFA-protein mRNA expression in developing bovine brain by in situ hybridization and northern blot hybridization.

Basic and applied histochemistry Pub Date : 1990-01-01
K Nakanishi, T Kitamura, M Okuda, T Mazaki, S Watanabe, N Miyoshi, S Fujita, M Fukuda
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Abstract

Using bovine brains of adult and developmental stages, the time and place of the appearance of mRNA for glial fibrillary acidic protein (GFA-protein) were studied by in situ- and Northern blot-hybridization. Double-stranded cDNA labeled with [3H]dCTP or [32P]dCTP was used as the probe for this mRNA. To compare the location of GFA-protein mRNA and GFA-protein itself on serial sections. GFA-protein immunohistochemistry was used. By in situ hybridization with adult bovine brain. GFA-protein mRNA was detected in astroglia, most of which were in the white matter. The distribution of these astroglia by in situ hybridization was consistent with the findings by GFA-protein immunohistochemistry and Northern blot hybridization, indicating that each techniques were specific. Concerning fetal stages, GFA-protein mRNA could be detected in the brain of a fetal calf with a body length of 28 cm by in situ hybridization using the 32P-labeled probe, and the mRNA was localized in the subpial area and the fornix. These results indicated that glial maturation first became recognizable at least in the subpial area and the fornix in the brain of a fetal calf measuring 28 cm. In this fetal brain, GFA-protein mRNA was almost undetectable by Northern blot hybridization. This suggested that in situ hybridization was more sensitive and useful for the analysis of gene expression than Northern blot hybridization, when the target mRNA is present in only a limited area, such as in the brain.

原位杂交和northern blot杂交分析发育中的牛脑gfa蛋白mRNA的表达。
采用原位杂交和Northern杂交技术,研究了成年和发育阶段牛脑胶质纤维酸性蛋白(GFA-protein) mRNA出现的时间和地点。用[3H]dCTP或[32P]dCTP标记的双链cDNA作为该mRNA的探针。比较gfa -蛋白mRNA和gfa -蛋白本身在序列切片上的位置。采用gfa蛋白免疫组化。通过与成年牛脑原位杂交。星形胶质细胞中检测到gfa蛋白mRNA,其中大部分位于白质中。这些星形胶质细胞的原位杂交结果与gfa蛋白免疫组织化学和Northern blot杂交结果一致,表明每种技术都具有特异性。在胎期,32p标记探针原位杂交可在体长28 cm的胎犊脑组织中检测到gfa蛋白mRNA, mRNA定位于颅底下区和穹窿。这些结果表明,至少在28厘米的胎犊大脑的枕下区和穹窿中,胶质细胞首先成熟。在这个胎儿大脑中,gfa蛋白mRNA几乎无法通过Northern blot杂交检测到。这表明,当目标mRNA仅存在于有限区域(如大脑)时,原位杂交比Northern blot杂交更敏感,更适用于基因表达分析。
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