Zen 3.3 blue edition software

EUFA1341 stable cells were seeded on glass-bottom dishes at 100,000 cells per dish. The next day, cells were irradiated with 10 Gy and processed for immunofluorescence after 6 h of recovery. Cells were washed with PBS and fixed in 4% paraformaldehyde for 15 min at room temperature. Following PBS washing, cells were permeabilized with 0.1% Triton X-100 for 5 min and then incubated in blocking solution containing 1% BSA for 1 h. Cells were then incubated with primary antibodies anti-BRCA1 (Santa Cruz Biotechnology Cat# sc-6954, 1:100, Dallas, TX, USA), anti-RAD51 (Abcam Cat# ab133534, 1:1000, Cambridge, UK), anti-FLAG (Sigma Cat# F1804, 1:1000, St. Louis, MO, USA), and anti-γH2AX (Abcam Cat# ab81299, 1:250, Cambridge, UK) overnight at 4 °C. After PBS washing, cells were incubated with second antibodies Alexa Fluor 488 goat anti-mouse (Abcam Cat# ab150113, 1:500, Cambridge, UK) and Alexa Fluor 647 goat anti-rabbit (Abcam Cat# ab150079, 1:500, Cambridge, UK) for 1 h at room temperature. Nuclei were stained for 10 min with 4′,6-diamidino-2-phenylindole (DAPI) before analysis. Images were captured using a ZEISS LSM 710 microscope, and the ZEN 3.3 (blue edition) software (ZEISS, Oberkochen, GER) was used for RAD51 foci analysis.