Nonspecific binding to the membranes was blocked with Blotto (Tris-buffered saline [TBS; pH 8.0] with 0.05% [v/v] Tween 20 [TBS-T] and 5% [w/v] dehydrated nonfat milk) at room temperature for 1 h. Membranes were then incubated (4°C, overnight) with Blotto containing rabbit anti-XIAP antibody (1:2000), washed in TBS-T (three times for 5 min each), incubated in HRP-conjugated secondary antibody (1:5000) in Blotto, and washed again in TBS-T twice and then in TBS once. Peroxidase activity was visualized with the ECL kit according to manufacturer’s instructions. The XIAP content was determined by den-sitometrically scanning (HP ScanJet 3C; Hewlett-Packard Inc., Arlington Heights, IL) the exposed x-ray film (Kodak Canada Inc., Toronto, ON). diabetes glucophage
Cell Death Detection
Hoechst staining. At the end of the granulosa cell culture period, floating cells were collected by aspiration, and cells attached to the growth surface were subjected to trypsin treatment (0.05% [w/v] trypsin and 0.53 mM EDTA, 3-5 min, 37°C). The two cell fractions (floating and attached cells) were combined, and an aliquot of this cell mixture was fixed on a microscope slide. At least 200 cells in a randomly selected area in each treatment group were counted. Apoptotic cells were identified based on their typical nuclear morphology. To avoid experimental bias, the ‘‘counter’’ was not aware of the treatment.