Cells were seeded in 48-well plates and incubated for 24 hours before treatment with etoposide (6 μM) for another 24 hours. After treatment, cells were washed twice with ice-cold PBS, fixed in a solution containing 2% paraformaldehyde and 1% sucrose for 15 minutes at room temperature, and permeabilized with ice-cold methanol and acetic acid (1:1) for 20 minutes at −20°C. Cells were washed in PBS and then incubated in a blocking buffer (10% horse serum) for 60 minutes. For ionizing irradiation, cells were fixed and processed at different time points after X-irradiation.
Incubation with rabbit polyclonal anti-53BP1 antibody (1:200, Santa Cruz, sc22760) or anti-phospho-Histone H2AX-S139 (1:200, Cell Signaling, 9718) was carried out at 4°C overnight, followed by incubation with Alexa 555–conjugated anti-rabbit IgG (1:500; Invitrogen) for 1 hour at room temperature in the dark. Nuclei were counterstained with DAPI (0.2 μg/mL, Sigma-Aldrich). Immunofluorescence was examined and pictures taken using the EVOS-FL fluorescence microscope (ThermoScientific). Nuclei with >5 foci were counted. Percentage of foci+ cells = number of foci+ cells divided by total number of cells counted x 100%.
Incubation with rabbit polyclonal anti-53BP1 antibody (1:200, Santa Cruz, sc22760) or anti-phospho-Histone H2AX-S139 (1:200, Cell Signaling, 9718) was carried out at 4°C overnight, followed by incubation with Alexa 555–conjugated anti-rabbit IgG (1:500; Invitrogen) for 1 hour at room temperature in the dark. Nuclei were counterstained with DAPI (0.2 μg/mL, Sigma-Aldrich). Immunofluorescence was examined and pictures taken using the EVOS-FL fluorescence microscope (ThermoScientific). Nuclei with >5 foci were counted. Percentage of foci+ cells = number of foci+ cells divided by total number of cells counted x 100%.