Saber M A, Zern M A, Shafritz D A
Proc Natl Acad Sci U S A. 1983 Jul;80(13):4017-20. doi: 10.1073/pnas.80.13.4017.
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.
我们提出了一种简单且改进的方法，用于在分离的小鼠肝细胞中原位定位白蛋白和胶原蛋白mRNA。通过胶原酶灌注、切碎和差速离心来分离细胞。然后，将经缺口平移的3H标记小鼠白蛋白cDNA（pmalb-2）和鸡原α2（I）型胶原蛋白cDNA（pCg45）探针与硅烷处理的微量离心管中的细胞进行杂交。将细胞转移并固定到载玻片上，通过计数置于细胞上的放射自显影片乳剂中的颗粒曝光量来半定量评估杂交情况。用这种原位杂交方法，所有肝细胞似乎都有大量但高度可变的白蛋白mRNA。此外，I型前胶原mRNA在肝细胞中的丰度似乎较低。这些结果表明，原位杂交可以有效地证明在分离的、充分分化的真核细胞中存在特定的低丰度或高丰度mRNA。
