AUTOMATED DOUBLE-LABELING OF PROLIFERATION AND APOPTOSIS IN GLUTATHIONE S-TRANSFERASE-POSITIVE HEPATOCYTES IN RATS

Immunohistochemical markers for proliferation (bromodeoxyuridine, BrdU ) and apoptosis (in situ terminal deoxynucleotide transferase dUTP nic k end-labeling, TUNEL) were localized within glutathione S-transferase (GSTP)-positive hepatic foci in rats. Using the TechMate Automated St aining System (BioTek Solutions; Santa Barbara, CA), formalin-fixed, p araffin-embedded sections were run through a double-label avidin-bioti n-immunoperoxidase protocol in less than 10 hr. Steam heat-induced epi tope retrieval and/or pro-automation teolytic digestion preceded each labeling procedure. Color development was achieved using diaminobenzid ine (DAB) with nickel enhancement for BrdU and TUNEL and VIP for GSTP. Results illustrate clear staining, brown-black BrdU-positive nuclei o r TUNEL-positive apoptotic bodies within purple GSTP-positive hepatocy tes. This automated procedure provides a method to easily identify and quantitate proliferating or apoptotic cells within foci of altered he patocytes in rat liver and may have general applications for studies o f cell or tissue kinetics during development, differentiation, and var ious pathological conditions in animals and humans.