To prepare protein extracts, cell pellets were lysed by heating (95°C, 5 min) in SDS sample buffer (50 mM Tris [pH 7.5], 5% SDS, 5 mM DTT, 5% glycerol) containing 6 M urea and were cleared by centrifugation (14 000 X g, 20 min). Supematants were analyzed for protein content using the Bio-Rad DC protein assay and stored at — 80°C until use. Protein extracts resolved by SDS-PAGE using the BioRad Miniprotean electrophoresis system were electrotransferred (30 V, 16 h) to nitrocellulose membranes. The membranes were then blocked (room temperature, 1 h) with blotto (TBS-T [Tris-buffered saline, 0.05% Tween-20] with 5% milk powder), then incubated (room temperature, 2 h) with primary antibody, washed in TBS-T (3X5 min), incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:10 000) in blotto, and washed again in TBS-T. Peroxidase activity was visualized with the ECL kit, according to the manufacturer’s instructions. buy ortho tri-cyclen online
Localization of Cell Death and Caspase-3 in Ovarian Sections
Ovaries fixed in 10% neutral buffered formalin were dehydrated through a graded series of ethanol (70-100%), cleared in xylene, embedded in paraffin, and sectioned (4 (xm). The sections were mounted on AES-coated slides, cleared in xylene, and then rehydrated through a graded series of ethanol (100-70%) to PBS. Terminal deoxynu-cleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) was performed with fluorescein-conjugated nucleotide to localize cell death in ovarian sections as previously described. To localize caspase-3 in adjacent histological sections, rehydrated ovarian sections were blocked in 5% BSA for 10 min, and then incubated (3 h, 37°C) in 5% BSA containing 5 fxg/ml goat anti-rat CPP32 (caspase-3) antibody or 5 |xg/ml normal goat serum (control). Slides were then washed in PBS (3 X 10 min), and immunostain-ing was visualized using an anti-goat IgG biotin-streptavi-din-HRP detection system, according to manufacturer’s instructions. Sections were dehydrated through a graded series of ethanol (70-100%) to xylene and mounted for examination.