DNA double-strand break assay by immunofluorescence staining

DNA double-strand breaks were studied using anti-γH2AX immunofluorescence staining. Briefly, HUVECs were grown on chamber slides and treated as described above. Cells were fixed in cold methanol for 10 min, washed with PBS three times, blocked using 1% BSA for 1 h at room temperature and incubated with primary anti-γH2AX antibody (1:200 dilution) at 4°C overnight. Following washing, cells were incubated with secondary Alexa Fluor 647-conjugated anti-mouse antibody (1:200 dilution) for 1 h at room temperature. Following rinsing, the nuclei were fluorescently labelled with DAPI in mounting medium. The images were examined under a fluorescence microscope.