Immunostaining of 53BP1 DNA Damage Foci

For the 53BP1 immunostaining experiment, 5000 cells/well were seeded in a black, transparent bottom 96-well plate. After incubation for 24 h with digested and non-digested samples, cells were fixed with 4% formaldehyde, pH 7.4, permeabilized with 0.2% Triton X-100 and washed three times with PBS containing 3% Bovine Serum Albumine (BSA) (washing buffer). They were then exposed to anti-53BP1 antibody (Abnova, PAB12506, dilution 1/1000, Taipei, Taiwan) for 1 h at room temperature under mild agitation, rinsed three times for 5 min with washing buffer, and incubated for 1 h at room temperature with goat anti-rabbit IgG-Atto 488 (Sigma Aldrich 18772, dilution 1/2000, St. Louis, MO, USA). They were further rinsed three times in washing buffer and counterstained with 0.3 µg/mL Hoechst 33,342 for 20 min at room temperature. Cells were finally washed three times with PBS, and plates were stored at 4 °C in the dark until analysis using a CellInsight CX5 High Content Screening platform (Thermo Fisher Scientific, Waltham, MA, USA), as reported previously [35 (link)].