Mouse anti p21 p1484

Immunostaining was performed as previously described (Jin & Kim 2017) (link). The antibodies used were rabbit anti-RAD51 (sc-8349, 1:100; Santa Cruz Biotechnology), mouse anti-γH2AX (ab26350, 1:100; Abcam), rabbit anti-ATM (pS1981, 1:100; Cell Signaling Technology), goat anti-OCT4 (sc-8628, 1:100; Santa Cruz), rabbit anti-p53 (sc6243, 1:100; Santa Cruz), rabbit anti-SOX2 (sc-17320, 1:100; Santa Cruz), and mouse anti-p21 (P1484, 1:1000; Sigma-Aldrich). The embryos were washed three times with phosphate-buffered saline (PBS)-PVA, and then labeled with a FITC-conjugated antibody (1:100) for 1 h at room temperature. The embryos were counterstained with 5 μg/mL Hoechst 33342 (Sigma Life Science) for 15 min, mounted on a glass slide and examined using an LSM 710 META confocal laser-scanning microscope (Zeiss).

A total of 300 porcine embryos were placed in 1× SDS sample buffer and heated at 99°C for 5 min. Proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes in 1× transfer buffer. Thereafter, membranes were blocked in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) containing 5% nonfat milk for 1 h and were then incubated at 48°C overnight with rabbit anti-p53 (sc6243, 1:500; Santa Cruz), mouse anti-p21 (P1484, 1:1000; Sigma-Aldrich) or rabbit anti-β-actin (13E5, 1:1000; Cell Signaling Technology). Membranes were washed three times with TBS-T (10 min each) and incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit-IgG (1:1000; Santa Cruz Biotechnology). Signals were detected using Pierce ECL Western blotting substrate (Thermo Fisher Scientific). To quantify Western blot results, band intensity values were determined using ImageJ software.