Abstract
In situ hybridization using radioisotope-labeled cDNA probe was performed on cow brain. The cDNA fragment for glial fibrillary acidic protein (GFA-pro-tein) mRNA of cow was labeled with 32P-dCTP or 3H-dCTP by nick translation or random priming, and used as the probe. X-ray film macroautoradiograph of in situ hybridization using 32P-labeled probe revealed that the positive signals for GFA-protein mRNA were mainly located in the white matter of cow brain. By in situ hybridization using 3H-labeled probe, silver grains indicating existence of GFA-protein mRNA were detected on the cytoplasm of astroglia, but not on the nerve cells, vascular endothelial cells, choroid plexus and pia mater. The backgrounds were negligibly small. Specific grains were not observed on the control sections. These results were consistent with the findings of GFA-protein immunohistochemistry and Northern blot hybridization, indicating satisfactory specificity of in situ hybridization by the protocol presented here. The critical analysis of the specificity of hybridization-singles was thought to be the most important prerequisite in interpreting the result of in situ hybridization.
