Automated double labeling of proliferation and apoptosis in glutathione S-transferase-positive hepatocytes in rats

Abstract

Immunohistochemical markers for proliferation (bromodeoxyuridine, BrdU) and apoptosis (in situ terminal deoxynucleotide transferase dUTP nick end- labeling, TUNEL) were localized within glutathione S-transferase (GSTP)- positive hepatic foci in rats. Using the TechMate Automated Staining System (BioTek Solutions; Santa Barbara, CA), formalin-fixed, paraffin-embedded sections were run through a double-label avidin-biotin-immunoperoxidase protocol in less than 10 hr. Steam heat-induced epitope retrieval and/or proteolytic digestion preceded each labeling procedure. Color development was achieved using diaminobenzidine (DAB) with nickel enhancement for BrdU and TUNEL and VIP for GSTP. Results illustrate clear staining, brown-black BrdU- positive nuclei or TUNEL-positive apoptotic bodies within purple GSTP- positive hepatocytes. This automated procedure provides a method to easily identify and quantitate proliferating or apoptotic cells within loci of altered hepatocytes in rat liver and may have general applications for studies of cell or tissue kinetics during development, differentiation, and various pathological conditions in animals and humans.