The slides were immersed in a blocking solution (5% nonfat skim milk in PBS) in a moisture chamber at 25°C for 1 h and washed with PBST (0.05% Tween in PBS) four times, each for 15 min. The testis sections on slides were preincubated with 3.0 ^M P12 in PBS at 25°C for 1 h, and the slides were washed with PBST as above. The slides prepared from each tissue were incubated for 2 h in the presence of the P12-induced antiserum diluted 1: 250 in blocking solution. After the slides had been gently agitated in four changes of PBST for 15 min each, the antibody to P12 were immunodetected with alkaline phosphatase-conjugated anti-rabbit IgG (Sigma, St. Louis, MO) diluted 1:1000 in the blocking solution. The sections were covered with 50% glycerol in PBS and photographed with a microscope (AH3-RFCA; Olympus, Tokyo, Japan) after they had been washed with three changes of PBS for 15 min each. birth control yasmin
Spermatozoa were air-dried on a glass slide and washed twice with PBS before cytochemical staining. The slides were immersed in 3.5% perchloric acid containing 0.04% Coomassie G-250 to manifest the intact acrosomes of spermatozoa according to previously described methods. To examine P12-binding region on spermatozoa, the slides were incubated with 3.0 ^M P12 for 40 min, washed with PBS, and incubated with the P12-induced antiserum diluted 1:250 in the blocking solution for 30 min. The slides were washed three times with PBS to remove excess antibody before they were incubated with fluorescein-conjugated goat anti-rabbit IgG (Sigma) diluted 1:100 in the blocking solution for 50 min. All of the slides were rinsed with PBS and covered with PBS:glycerol (1:1 by volume) before observation under a microscope equipped with epi-fluorescence (AH3-RFCA).