On the basis of daily ultrasonographic records, follicles were identified as the dominant, largest subordinate, and second-largest subordinate on the day of ovariectomy. Regarding the dominant and largest subordinate follicles, only those that were individually identified were collected and analyzed. If on the day of ovariectomy the second-largest subordinate follicle could not be distinguished from other subordinates (i.e., similar diameter; D6W1, Da 17), one was chosen arbitrarily and included in the second-largest subordinate category. Hence, the second-largest subordinate category included follicles that were individually identified as well as those that could not be individually identified. On D1W2, the three largest follicles of wave 2 were collected to represent preselection follicles because dominance was not yet apparent. Cheap Diskus Advair
Polyclonal rabbit antiserum (Rb-32, bleed date 02-2390) raised against native porcine follistatin was generously provided by Dr. Nicholas Ling (Whittier Institute for Diabetes and Endocrinology, La Jolla, CA) and was used as the primary antibody for immunohistochemical localization of follistatin. The antiserum has been characterized and does not cross-react with activin A or inhibin A. The binding ability of antiserum was tested using Western blotting, and it detected recombinant human follistatin (rhFS-288; Code #B3904 National Hormone and Pituitary Program, NIDDK, Rockville, MD) and recombinant porcine follistatin (gift from C.R. Christensen, University of Saskatchewan, Saskatoon, SK, Canada). Lyophilized primary antiserum was stored at -20°C until use and diluted 1:1000 with 4% normal goat serum prepared in PBS (0.1 M phosphate buffer, 0.9% NaCl, pH 7.2-7.4). Excess of rhFS-288 was added to half of the diluted primary antiserum (10 mg rhFS-288/ml of 1:1000 diluted antiserum) and allowed to stand overnight at 4°C to adsorb the primary antibody. The adsorbed and nonadsorbed antisera were then centrifuged for 15 min at 3000 X g to remove any particulate material. Follistatin-adsorbed antiserum was used to measure the degree of nonspecific staining for quantitative immunohistochemistry.