Immunofluorescence staining analysis of γH2AX and RAD51

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.