All bovine tissue was collected at a local abattoir, within 20 min of slaughter, from cows assessed visually as being nonpregnant. A slice of up to 5 mm was cut through the center of ovaries (n = 10) to be used for immunohisto-chemistry, and the slices were immediately immersed in Tissue-Tek OCT embedding compound (Miles Inc., Elkhart, IN) and snap-frozen; these blocks were stored at -70°C. Tissue sections (10 ^m) were cut using a CM1800 Leica (Milton Keynes, Bucks, UK) cryostat, collected on glass slides freshly treated with 0.01% poly-L-lysine hydrobromide (cat. #P-1524; Sigma Chemical Co., St. Louis, MO) or 0.01% poly L-ornithine hydrobromide (cat. #P-4638; Sigma), and stored at – 20°C until use. For Western blot analyses, tissues were placed into Hepes-buffered Earle’s balanced salt solution without calcium or magnesium and placed on ice during transport to the laboratory. buy yasmin online
Antral follicles (3-10 mm) were dissected from the ovaries, and all adhering surface epithelium was removed. Follicles were pooled on the basis of diameter into four categories: < 3 mm (n = 10 follicles); 3-5 mm (n = 9 follicles); 510 mm (n = 7 follicles); > 10 mm (n = 4 follicles). The follicular fluid was gently removed by aspiration, and the remaining tissue of each follicle was weighed (pooled weights = 30 mg, 109 mg, 304 mg, 527 mg for categories 1-4, respectively) and stored at – 20°C until required. Pieces of two kidneys were similarly weighed and stored.