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Increased Apoptosis Occurring During the First Wave of Spermatogenesis: MATERIALS AND METHODS(5)

METHODS(5)

Subsequently, the slides were incubated with primary rabbit polyclonal antibodies directed against Bcl-2 (Upstate Biotechnology) or against Bax or Bad (Santa Cruz Biotechnology) at dilutions of 1:400, 1:200, and 1: 200, respectively, in PBS containing 0.1% goat serum for 60 min. After extensive washing in PBS-T, these slides were then incubated for 30 min with a secondary biotinylated goat anti-rabbit immunoglobulin G (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1:1000 in PBS containing 0.1% goat serum. After five additional washes with PBS-T, the sections were incubated with avidin-conjugated horseradish peroxidase (Vector Laboratories, Burlingame, CA). In the case of 18- and 26-day-old rats, adjacent sections were also stained with Mayer hematoxylin to allow accurate identification of the different stages of the seminiferous epithelial cycle. Sections of rat thymus and brain treated in this same manner served as positive controls. Negative-control sections were treated as described above, except that instead of being incubated with a primary antibody, normal serum from appropriate animal species was employed instead. More info

Statistical Analysis

In the case of vital cell preparations and TUNEL staining, at least 10 replicate samples from each of four rats in each group were examined. The corresponding number for the electron-microscopic investigation was three samples from each animal; for immunoblotting, the total protein fraction from each animal; and for immunohistochemical analysis, three samples from each animal. The quantitative data in the figures are presented as the mean ± SEM, whereas the nonparametric data in the table are presented as median values together with the 25% and 75% percentiles. The Mann-Whitney Latest was employed for comparison of independent groups of samples, and the Kruskall-Wallis analysis with the Dunn post-hoc test was performed for multiple comparison of independent groups of samples. A P value of less than 0.05 was considered to indicate a statistically significant difference.