Immunohistochemical Staining for Follistatin
A pair of glass slides containing adjacent sections of tissue were incubated overnight with 1:1000 dilution of primary antiserum and follistatin-adsorbed primary antiserum (one slide each) to localize the follistatin and nonspecific reaction, respectively. The avidin-biotin peroxidase technique was used as described previously using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Microscope slides of all follicles under study were stained as a single batch using a Code-On-Histomatic slide-staining station (Fisher Scientific Co., Edmonton, AB, Canada) to minimize the variation in reaction due to difference in staining batches. Buy Advair Diskus Online
Similarly, all corpora lutea were stained as a single batch. Briefly, endogenous peroxidase was blocked with 2% v:v hydrogen peroxide in methanol for 10 min, and proteolytic enzyme digestion was done with 0.05% w: v protease XIV (catalogue no. P5147; Sigma Chemical Company, St. Louis, MO) for 30 min. Slides were treated with 4% normal goat serum (Gibco BRL, Life Technologies, Gaithersburg, MD) containing 0.05% v:v Tween 20 (Aldrich Chemical Co., Milwaukee, WI) for another 30 min to minimize nonspecific binding of the secondary antibody.