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Increased Apoptosis Occurring During the First Wave of Spermatogenesis: MATERIALS AND METHODS(4)

These membranes were then incubated with primary rabbit polyclonal antibodies directed against Bad, Bax, caspase 3, and PARP (diluted 1: 2000; Santa Cruz Biotechnology, Santa Cruz, CA) or against Bcl-2 (diluted 1:3500; Upstate Biotechnology, Lake Placid, NY) in TBS at room temperature for 1 h, washed five times with TBS containing 0.1% Tween-20 (PBS-T), and thereafter incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (diluted 1:104; Transduction Laboratories, San Diego, CA). The antigen-antibody complexes thus formed were quantitated by densitometry of the chemiluminescence emitted. After development of the films, the immunoblots were stained with Coomassie blue to confirm equal protein loading. Total testicular protein from each of the four rats in each group was immunoblotted in this manner.

Immunohistochemical Analysis

For immunohistochemical analysis, paraffin sections (thickness, 5 |xm) of formalin-fixed testes were first dewaxed in xylene and subsequently rehydrated by immersion in a series of baths containing decreasing concentrations of ethanol. Antigen retrieval was achieved by incubation in 0.01 M sodium citrate (pH 6.0) at 95-98°C for 20 min, after which the slides were washed in PBS and endogenous peroxidase activity quenched by incubation with 3% H2O2 in PBS for 10 min. Nonspecific binding was blocked by incubation with 1% untreated goat serum for 1 h. read