Use of in situ hybridization to identify collagen and albumin mRNAs in isolated mouse hepatocytes.
Use of in situ hybridization to identify collagen and albumin mRNAs in isolated mouse hepatocytes.
复制标题
使用原位杂交鉴定分离的小鼠肝细胞中的胶原蛋白和白蛋白 mRNA。
DOI：
10.1073/pnas.80.13.4017
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发表时间：
1983
影响因子：
11.1
通讯作者：
Shafritz,DA
中科院分区：
文献类型：
--
作者：
Saber,MA;Zern,MA;Shafritz,DA
我们提出了一个简单的和改进的方法，在原位定位的白蛋白和胶原蛋白的mRNA在离体小鼠肝细胞。通过胶原酶灌注、切碎和差速离心分离细胞。然后将缺口翻译的3 H标记的小鼠白蛋白cDNA（pmalb-2）和鸡前α 2（I）胶原cDNA（pCg 45）探针与硅烷处理的微量离心管中的细胞杂交。将细胞转移并固定在显微镜载玻片上，通过计数置于细胞上的放射自显影乳剂中的颗粒暴露量来半定量评价杂交。采用这种原位杂交方法，所有肝细胞似乎都具有显著但高度可变的白蛋白mRNA量。此外，I型前胶原mRNA似乎以低丰度存在于肝细胞中。这些结果表明，原位杂交可以有效地证明存在特定的低或高丰度的mRNA在分离的分化良好的真核细胞。
We present a simple and improved method for in situ localization of albumin and collagen mRNAs in isolated mouse hepatocytes. The cells were isolated by collagenase perfusion, mincing, and differential centrifugation. Nick-translated 3H-labeled mouse albumin cDNA (pmalb-2) and chicken pro-alpha 2(I) collagen cDNA (pCg45) probes were then hybridized with the cells in silane-treated microcentrifuge tubes. The cells were transferred and fixed to a microscope slide and hybridization was evaluated semiquantitatively by counting exposure of grains in autoradiographic emulsion placed over the cells. With this method of in situ hybridization, all hepatocytes appear to have significant, but highly variable, amounts of albumin mRNA. In addition, type I procollagen mRNA appears to be present at low abundance in hepatocytes. These results indicate that in situ hybridization can effectively demonstrate the presence of specific low- or high-abundance mRNAs in isolated well-differentiated eukaryotic cells.