Immunofluorescent staining for γ-H2AX

The DNA damage was evaluated by performing immunofluorescent staining for γ-H2AX. The HEI-OC1 cells were grown on polylysine-coated cover glasses. After radiation, the cells were fixed in 4% paraformaldehyde, permeabilized in Triton X-100 and blocked in 1% goat serum. Subsequently, the cells were incubated with the primary antibody against γ-H2AX (1:100 dilution; Abcam, San Francisco, CA, U.S.A.) overnight at 4°C, followed by incubation with Alexa Flour 488 secondary antibody (1:200 dilution; Abcam, San Francisco, CA, U.S.A.) for 1 h at room temperature in the dark. After being washed three times in PBS, cells were stained with DAPI and observed using a fluorescence microscope (Olympus, Tokyo, Japan).