Rabbit anti rad51 polyclonal antibody

Ovarian cancer cells were cultured on coverslips in 24-well plates for 48 hours in respective medium containing inhibitor. Cells were fixed with 4% paraformaldehyde, and blocked with a 5% BSA-phosphate buffer solution. The cells were then incubated with rabbit anti-RAD51 polyclonal antibody (Santa Cruz Biotechnology) or rabbit anti-γH2AX^Ser139 polyclonal antibody (Cell Signaling Technology) overnight at 4°C. After washing with PBS, the cells were incubated with secondary antibodies and DAPI at room temperature. Images were acquired and quantified using an immunofluorescence microscope (Leica). The dynamics of γH2AX and RAD51 foci accumulation, as well as percentage of positive cells (more than 5 foci in one cell) were calculated based on analysis of about 200 cells.

The cells were seeded onto sterile confocal dishes and exposed to a medium containing 3 μM olaparib, or PBS, for 24 h. The cells were fixed in pre-chilled methanol/acetone (7:3) at −20°C for 10 min. Subsequent to air-drying, the dishes were washed three times with PBS and blocked using 5% skimmed dry milk and 0.1% Triton X-100 in PBS at room temperature for 1 h. Samples were then incubated overnight at 4°C with mouse anti-phospho-Histone H2AX (Ser139) monoclonal antibody (Millipore, Billerica, MA, USA: dilution, 1:50) and rabbit anti-RAD51 polyclonal antibody (Santa Cruz, Dallas, TX, USA: dilution, 1:50) (11 (link)). Subsequent to being washed, the cells were incubated with secondary Cy3-labeled goat anti-mouse immunoglobulin G (IgG), and Alexa Fluor 488-labeled goat anti-rabbit IgG (H+L) antibodies (Beyotime, Suzhou, China), for 1 h at room temperature and protected from light. Subsequent to being washed again, the nuclei were stained with 1 μg/ml DAPI (Beyotime) for 10 min. Images were obtained with a confocal laser scanning microscope (Leica TCS SP8; Leica, Wetzlar, Germany).