Biotinylated goat anti chicken igg h l

Liver pieces were fixed in Z-Fix Concentrate (Anatech Ltd., Battle Creek, MI) for 24 hours at room temperature (RT), embedded in paraffin, and cut into 3 μm thick sections. The sections were deparaffinized in xylene and dehydrated in graded alcohol concentrations. Sections were treated with 0.3% hydrogen peroxide in methanol for 30 minutes to block endogenous peroxidase activity, incubated in avidin and biotin (Biotin Blocking System, DAKO, Carpinteria, CA) for 10 minutes each at RT, and then incubated sequentially with BLOKHEN II blocking solution (Aves Lab, Tigard, OR) for 20–30 minutes at RT and a chicken anti-mouse Fah antibody (a gift from Markus Grompe) at 4°C overnight. The sections were then incubated with biotinylated goat anti-chicken IgG (H+L) (Vector Laboratories, Burlingame, CA) and then an avidin-biotin-peroxidase complex (ABC/HPR; DAKO) for 45–60 minutes at RT. The color reaction was developed with 3,3′-diaminobenzidine tetrahydrochloride (DAB). Fah⁺ cells exhibited brown-stained cytoplasm after this procedure. To detect BrdU, the sections were then treated with a monoclonal rat anti-BrdU antibody (Abcam, Inc., Cambridge, MA) at 4°C overnight, biotinylated rabbit anti-rat IgG (H+L) (Vector Laboratories) for 45–60 minutes at RT, and then DAB (SK-4100 kit, Vector Laboratories). BrdU⁺ cells exhibited gray-black nuclei after this procedure.