NONRADIOACTIVE IN-SITU HYBRIDIZATION FOR DETECTION OF MESSENGER-RNAS ENCODING FOR PEROXISOMAL PROTEINS - HETEROGENEOUS HEPATIC LOBULAR DISTRIBUTION AFTER TREATMENT WITH A SINGLE-DOSE OF BEZAFIBRATE

We present a nonradioactive in situ hybridization (ISH) protocol for d etection of mRNAs in rat liver encoding for three peroxisomal proteins : catalase and urate oxidase as representatives of high-level abundanc e mRNAs and trifunctional protein (PH) as that of low-level abundance mRNAs. In addition to normal rats, animals treated for 24 hr with a si ngle dose of bezafibrate were studied. The use of perfusion-fixation w ith 4% depolymerized paraformaldehyde/0.05% glutaraldehyde combined wi th paraffin embedding and the application of digoxigenin-labeled cRNA probes provided optimal cytological resolution and high sensitivity co mparable to that of radioactive ISH, In parallel experiments, the same digoxigenin-labeled cRNA probes were used for Northern and semiquanti tative doe-blot analysis of isolated RNAs. In control animals, the mRN As for catalase and urate oxidase were uniformly distributed across th e liver lobule and were confined to liver parenchymal cells. The bile duct epithelial and the sinusoidal cells remained negative. The specif icity and the high resolution of our protocol were further substantiat ed by reciprocal localization of transcripts for albumin and glycerald ehyde-3-phosphate dehydrogenase in different regions of the liver lobu le and for catalase in the proximal tubules of the renal cortex, Where as in control livers the transcripts for PH were barely detectable, a strong signal was found in pericentral hepatocytes of bezafibrate-trea ted animals, corresponding to an 8-10-fold increase of mRNA detected i n dot-blots, In contrast, the urate oxidase mRNA was reduced by more t han 50%, with diminution of staining in pericentral regions of the liv er lobule, The mRNA encoding for catalase was only slightly affected, Further applications of this protocol should be helpful in elucidation of the cell-specific transcriptional regulation of peroxisomal protei ns in various organs under normal and pathological conditions.