Gerdes, 2000). With the exception of the DNA denaturing step for BrdU, assays followed the same basic protocol as follows: cryoprotectant was rinsed off in tris buffered saline (TBS, Bio-Rad, Hercules, CA) and endogenous peroxidases were quenched by 0.3% H2O2 in TBS. Following washes, sections were blocked in TBS+ (TBS/0.1% or 0.2% of triton-X/3% normal serum as appropriate) and incubated in primary antibody at +4°C for 16–48 h, dependent upon the antibody. Primary antibody dilutions and durations were determined in dilution curves and antibody specificity was verified by control experiments that omitted the primary antibody, secondary antibody and/or BrdU injection. After incubation, sections were washed in TBS+ and then incubated in the appropriate biotinylated secondary antibody plus 1.5% serum for 1 h, followed by avidin-biotin-peroxidase complex (ABC elite kit, Vector Labs) for 1 h, and Nickel-enhanced DAB (Polysciences, Warrington, PA) as a chromagen. Sections were mounted onto glass slides and dried. For Ki-67 labeled tissue, mounted sections were counterstained in neutral red dye (Fisher Scientific, Waltham, MA) whereas the BrdU labeled tissue was counterstained with cresyl violet dye (Fisher Scientific). All sections were coverslipped with Cytoseal® (Stephens Scientific, Wayne, NJ)
