Use of in situ hybridization to identify collagen and albumin mRNAs in isolated mouse hepatocytes.
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.
D000818:Animals; D003001:Cloning, Molecular; D003094:Collagen / Q000235:genetics*; D004247:DNA / Q000032:analysis; D066298:In Vitro Techniques; D008099:Liver / Q000378:metabolism*; D051379:Mice; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics*; D012709:Serum Albumin / Q000235:genetics*
D012333:RNA, Messenger; D012709:Serum Albumin; D003094:Collagen; D004247:DNA