Immunofluorescent staining and DNA repair foci analysis

OPSCC cells were grown on 13 mm coverslips until ~70–80 % confluent, irradiated at 4 Gy and incubated for the required time in 5 % CO₂ at 37°C to allow for DNA repair. Cells were washed with PBS at room temperature for 5 min before being fixed using 4 % paraformaldehyde for 10 min. Cells were permeabilised with 0.2 % Triton X-100 in PBS for 10 min, then washed three times with 0.1 % Tween-20 for 10 min. Coverslips were blocked to avoid non-specific staining via incubation with 2 % BSA for 30 min at room temperature on a rocking platform with either γH2AX, 53BP1 or RAD51 antibodies in 2 % BSA overnight at 4°C. Following three washes with PBS, coverslips were incubated with either goat anti-mouse Alexa Fluor 555 or goat anti-rabbit Alexa Fluor 488 secondary antibodies in 2% BSA for 1 h at room temperature in the dark. Finally samples were washed with PBS for 10 min on a rocking platform and mounted on a microscope slide using Fluoroshield containing DAPI (Sigma-Aldrich, Gillingham, UK). Cells were examined using an Olympus BX61 fluorescent microscope with a Photometrics CoolSNAP HQ2 CCD camera. MicroManager software was used to capture images (~500 images/cell line/antibody).