Horseradish peroxidase conjugated digoxigenin antibody

The apoptotic cell death was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. Cells treated with various concentrations of aqueous BJ extract for 12 hours were permeabilized in 10 mM citrate buffer, pH 6.0. After blocking the nonspecific labeling with PBS mixture containing 2% bovine serum albumin and 0.5% NP-40, cells were incubated in TUNEL reaction solution mixed with 9 mM dUTP, 1 mM digoxigenin-labeled dUTP (Roche, Mannheim, Germany), 2.5 mM cobalt chloride, 100 mM Tris pH 7.6 and 0.3 U/μL terminal deoxynucleotidyl transferase for 1 hour at 37°C in a humidified atmosphere. The sections were washed with PBS and incubated with a 1:200 dilution of horseradish peroxidase-conjugated digoxigenin antibody (Roche). With removal of unbound antibody, cell images were taken by fluorescence or confocal microscope. All colored TUNEL images were converted to black and white by Photoshop software, before being quantitated with Multi Gauge software (version 2.1, FUJIFILM). Sections of positively stained cells containing 50 nuclei were counted. Five slides of each concentration and control sections were recorded and three independent experiments carried out.