LIVER-REGENERATION - A COMPARISON OF IN-SITU HYBRIDIZATION FOR HISTONE MESSENGER-RNA WITH BROMODEOXYURIDINE LABELING FOR THE DETECTION OF S-PHASE CELLS

We developed an in situ hybridization technique for measurement of pro liferative cell numbers through detection of histone mRNA in routinely fixed, paraffin-embedded tissue sections, Histone gene expression is coordinated with the fell cycle, and the increase in expression during S-phase permits unambiguous identification of cells undergoing DNA re plication. Histone mRNAs were identified in routinely processed rat li ver tissue by non-isotopic in situ hybridization with digoxigenin-labe led oligonucleotide probes. Specific hybrids were detected with alkali ne phosphatase-labeled anti-digoxigenin antibody and visualized by BCI P-nitroblue tetrazolium indicator substrate. Unequivocal cytoplasmic l abeling was observed in various cell types in the liver remnant during the first 72 hr after a two-thirds partial hepatectomy. The spatial a nd temporal patterns of histone labeling were almost identical to thos e obtained by staining with an antibody to bromodeoxyuridine. The iden tification of histone mRNA appears to be a reliable marker of the S-ph ase fraction, a technique with the further advantage that the tissue d oes not have to be first exposed to a nucleotide analogue. Hence, retr ospective studies are possible. The probes can be applied to human and animal cells and tissues because the nucleotide sequences of histone genes are conserved.