Heifers were treated postoperatively with procaine penicillin G for 4 days. Immediately upon removal, the ovarian artery or its branches were cannulated, flushed with 15-20 ml PBS (0.1 M phosphate buffer, 0.9% sodium chloride, pH 7.27.4) to remove blood, and then perfused with 30 ml of Karnovsky’s fixative containing 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at a flow rate of 2 ml/min. The fixation procedure was completed within 45 min of ovariectomy. The ovaries were immersed in Karnovsky’s fixative for 3 h, after which each individually identified dominant follicle, subordinate follicle, and corpus luteum was sectioned through its largest diameter. Slices of follicles and corpora lutea were fixed for another 3 h by immersion in aqueous Bouin’s fixative for light microscopy.
The double-fixation procedure permitted use of the same tissues for light microscopy, scanning and transmission electron microscopy, and immuno-histochemistry. Initial perfusion fixation helped to maintain the structural integrity of individual follicles, thus preventing their collapse and artifactual loss of adluminal granulosa cells due to coagulation of follicular fluid. In addition, perfusion fixation resulted in retention of a thin layer of coagulated follicular fluid along the antral wall throughout tissue processing, thus permitting assessment of sloughed cells. Tissues were processed as described above to obtain 5-mm paraffin sections on poly-L-lysine-coated glass slides. In addition to follistatin immunohistochemical staining, adjacent sections were stained with hematoxylin and eosin to confirm the architecture of tissues under study.