DNA repair foci—immunofluorescence microscopy

After deposition on slides using a Cytospin centrifuge, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton in phosphate-buffered saline (PBS) and saturated with 5% bovine milk in PBS. The rabbit anti-53BP1 antibody (clone NB100-304, Novus Biologicals, Cambridge, UK) and the mouse anti-γH2AX (Ser139) antibody (clone JBW301, Merck Millipore, Darmstadt, Germany) were diluted 1/300 and 1/100, respectively, in 5% bovine milk in PBS, and deposited on cytospins for 90 min at room temperature. Slides were washed twice and antibodies to rabbit or mouse immunoglobulins conjugated to alexa 488 (diluted 1/500 in 5% bovine milk in PBS) were added for 45 min at room temperature. Slides were washed and mounted with Vectashield and 1% DAPI. Images and fluorescence were captured with a ZEISS Axio Imager Z2 microscope (× 63 objective), analyzed with Metafer (version3.6, company, town state) and ImageJ software. The number of 53BP1 and γH2AX foci was counted in at least 300 nuclei.

Supplementary informations concerning methodology are included in Supplementary experiment procedures.