Anti rabbit igg fitc conjugate

Both COCs and cumulus cells and zona pellucida removed oocytes were used for immunostaining. The cumulus cells of COC were removed by vibrating for 10 minutes. The zona pellucida was removed by acidic Tyrode solution treating. COCs and Oocytes were fixed in 4% paraformaldehyde for 40 minutes, and then permeabilized in PBS with 0.1% Triton X-100 for 20 minutes. Next, oocytes were blocked in PBS containing 1% BSA for 1 hour and incubated with γH2A.X antibody (Bioworld) and/or cAMP antibody (AM01, Santa Cruz Biotechnology) at 4°C overnight. After 3 washes with PBST, oocytes were incubated with secondary antibody (Anti-Rabbit IgG FITC conjugate, Sigma; Cy3 conjugated goat anti mouse IgG antibody, Beyotime) and washed 5 times with PBST. DNA was stained with Hoechst stain and the oocytes were mounted on slides. We used a confocal laser-scanning microscope to observe the oocytes.

Anthers were fixed for 30 min in 4% (w/v) para-formaldehyde (PFA) in PMEG buffer (50 mM PIPES, 1 mM MgSO_4, 5 mM EGTA, 1% glycerol, pH 6.8) [57 ]. After washing in PBS for 10 min, the anthers were digested for 15 min at 37°C in a mixture of 2% Cellulase Onozuka RS (Yakult Co. Ltd., Tokyo) and 5% Pectolyase Y23 (Seishin Kagaku, Tokyo). The anthers were rinsed in PMEG twice for 5 min each, and squashed on a glass slides in PMEG. After freezing the slides in liquid nitrogen, the coverslips were removed and the slides were air-dried. The slides were incubated for 15 min in a detergent solution (0.5% Triton X-100 in PBS). Slides were washed three times in PBS for 5 min each and blocked with 1% BSA in PBS for 20 min, followed by three PBS washes for 5 min each, and stored until immunostaining. Anti-dimethyl-Histone H3 (Lys9) rabbit immunoaffinity purified IgG (1:200) (Upstate Biotechnology Inc., VA, USA) was used as the primary antibody. Slides were incubated with the primary antibody in a humid dark box at 4°C overnight. After washing the slides in PBS three times for 5 min each, anti-rabbit IgG FITC conjugate (1:80, Sigma, MO, USA) was applied as the secondary antibody. These slides were incubated for 3 hours in a humid dark box at 37°C. After washing the slides in PBS three times for 5 min each, the chromosomes were counter-stained with 1 μg/mL DAPI in Vectashield (Vector Lab.).