Immunofluorescence Staining of 53BP1 and γH2AX

Cells were grown on coverslips, treated as indicated and fixed in 4% paraformaldehyde for 15 min at room temperature. The coverslips were washed twice with PBS, permeabilized for 5 min in 0.2% Triton X-100, followed by 20 min blocking in filtered 1% BSA in PBS at room temperature. Primary antibody incubations were performed at room temperature for 2 hours. Secondary antibody incubations were performed at room temperature for 30 min. The coverslips were washed twice with PBS, incubated with DAPI (0.5 μg/mL, ThermoFisher) in PBS for 10 min at room temperature, washed three times with PBS and mounted on slides using Mowiol-based mounting media. Primary antibodies used: anti-53BP1 (mouse, 1:10; Schultz et al., 2000 (link)); anti-H2AX Phospho S139 (rabbit, Cell Signaling, 1:500). Secondary antibodies used: Alexa Fluor 488 (goat anti-mouse, ThermoFisher, 1:500); Alexa Fluor 594 (goat anti-rabbit, ThermoFisher, 1:500). Images were acquired with a Zeiss Imager M2 AX10 microscope as described in the section describing detection of mitotic EdU foci.