Quantifying RAD51 Nuclear Foci Formation

For RAD51 nuclear focus formation analysis, cells were cultured on coverslips for 24 h and then irradiated or not with 4 Gy using a RS2000 generator (Radsource). After irradiation, cells were allowed to recover for 4 h. Cells were then washed with PBS, treated with CSK buffer (10 mM PIPES pH 7.0, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl₂, 0.7% Triton X-100) for 2 min at 4°C, washed with PBS and fixed with 4% paraformaldehyde for 10 min at 4°C before an additional fixation step of 2 min with glacial methanol. Coverslips were rinsed with PBS and blocked for 1 h in PBS containing 0.1% Triton X-100 and 5% BSA. Cells were stained using a rabbit anti-RAD51 serum (1:5000) [106 (link)] in 0.5% BSA PBS at room temperature for 1 h prior to incubation with AlexaFluor 594 or 488 anti-rabbit secondary antibodies (1:5000, Molecular Probes) in 0.5% BSA PBS. Coverslips were mounted onto slides with Vectashield mounting media containing DAPI (Vector laboratories) and images were obtained using a Zeiss Axio Imager Z2 microscope with a 63x oil immersion objective. Maximum-intensity projection images were generated to display foci in all sections and were analyzed using ImageJ software. Automatic counting was performed using FoCo [107 (link)] and validated manually.