Immunofluorescence staining analysis of γH2AX and RAD51

OVCA433, OVCAR5, and OVCAR8 cells were cultured on coverslips in 24-well plates in respective medium containing inhibitors for 48 hours. The cells were incubated with rabbit anti-RAD51 polyclonal antibody (Santa Cruz Biotechnology) or rabbit anti-γH2AX (Ser139) polyclonal antibody (Cell Signaling Technology), and then incubated with secondary antibodies and DAPI. Images were acquired and quantified using an immunofluorescence microscope (Leica). The dynamics of phosphorylated histone H2AX (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.