Immunostaining for DNA Damage Foci

For immunostaining, cells were grown on coverslips and irradiated via the 365-nm pulsed nitrogen laser (Spectra-Physics) or 8 Gy of IR. For the cells irradiated with the laser, the coverslip was placed in a dark box and the cells were allowed to recover at 37°C for 30 min. The cells were subsequently washed with ice cold PBS three times and then fixed via incubation with 4% paraformaldehyde at room temperature for 20 min. The cells were permeablized with PBS containing 0.5% Triton X-100 on ice for 10 min, blocked with PBS containing 5% normal goat serum overnight at 4°C and then incubated with the primary antibodies at room temperature for 1 h. After washing with cold PBS, cells were incubated with the second antibodies conjugated with Alexa 488 (Invitrogen, A11001) or Texas red (Invitrogen, T2767). Following IR with 8 Gy, focus formation of RPA and Rad51 was detected using fluorescent immunostaining at various time points as described in the figure legends and as previously described (30 (link)).