To estimate the number of corpora lutea (CL) per ovary, sections were used to count the number of CL without knowledge of treatment group. To avoid double counting, each CL was followed through consecutive sections to ensure that it was only counted once.
After dosing, ovaries were fixed in 4% paraformaldehyde for 24 h. After fixation, the tissues were dehydrated, embedded in Paraplast (VWR Scientific, Baltimore, MD), serially sectioned (5 |xm), and mounted on silane-treated (Sigma Aldrich, St. Louis, MO) glass slides. The sections were processed for immunolocalization of Bcl-2 and Bax using a commercially available polyclonal antibody against Bcl-2 (1:50 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) and a polyclonal antibody against Bax (1:50 dilution; Santa Cruz Biotechnology) as the primary antibodies. The primary antibodies were found to be specific for their respective protein in previous studies conducted in our laboratory. Furthermore, a titration of antibody concentration was conducted to ensure specificity in our tissues (dilutions 1:20-1:200).