A commercially available kit (ApopTag; Intergen, Purchase, NY) was used for the detection of 3′-OH DNA ends in the sections. After washing with distilled water three times for 10 min each, the sections were incubated in the equilibration buffer of the kit for 20 sec at RT. Then, sections were incubated at 37°C for 1 h in a moist chamber with 60 |xl of the working buffer containing terminal deoxynucleotidyl transferase, digoxigenin-11-dUTP The reaction was stopped by incubating the sections in a blocking buffer containing sodium citrate and NaCl at 37°C for 30 min.
After rinsing with PBS four times for 15 min each, sections were incubated with antidigoxigenin antibody conjugated to horseradish peroxidase at RT for 30 min. After incubation with the antibody, the peroxidase activity was examined by exposing the sections to a solution containing 0.05% dia-minobenzidine and 0.01% H2O2 in Tris buffer (pH 7.6) for 3-6 min at RT. The sections were counterstained with 1% methyl green. For control experiments, the enzyme incubation step was omitted.
Experiments were repeated a least three times, and representative chemiluminescence was first scanned using a Power Macintosh computer (G3; Apple Computer, Inc., Cupertino, CA) equipped with a ScanJet 6100C scanner (Hewlett-Packard Co., Greeley, CO). Quantification of the scanned images was performed according to the NIH Image version 1.61 software (National Institutes of Health, Bethesda, MD). Data are expressed as the mean ± SEM of three experiments. Statistical analysis was performed by one-way ANOVA using SPSS version 11.0 software (SPSS, Chicago, IL).