For immunostaining, cells were generally fixed in 2% formaldehyde and permeabilized as described earlier [13 (link)]. For immunostaining of Ku80 we followed the protocol of Britton, et al. [44 (link)] with some changes: Pre-extraction with CSK+R buffer was performed 3x instead of 2 × 3 min, fixation occurred for 15 min with 4% instead of 2% formaldehyde, and permeabilization was done for 10 min instead of 5 min with PBS, 0.2% Triton X-100. Primary antibodies for immunofluorescence staining were diluted in 1x PBS, 0.4% BSA: α53BP1 (Ab-1) (rabbit, Calbiochem, PC712, 1:500), αCENP-F (rabbit, Novus, NB500-101, 1:750), αCtIP (rabbit, Bethyl Laboratories, A300-488A, 1:100), αGeminin (mouse, clone 1A8, Novus Biologicals, H00051053-M01, 1:100), αγH2AX (Ser 139) (mouse, clone JBW301, Millipore, 05-636, 1:500), αKu80 (mouse, clone 111, Thermo Scientific, MA5-12933, 1:100), αRPA/P34 (mouse, clone 9H8, Sigma, R1280, 1:3000). To visualize the primary antibodies, secondary Alexa 488-, Alexa 568-, or Alexa 647-conjugated αmouse or αrabbit antibodies from donkey or goat were used (Invitrogen, Life Technologies, Sigma-Aldrich). DNA was counterstained with DAPI (AppliChem; 1 µg/mL). In the end, the samples were mounted in Slow Fade Diamond Antifade (Invitrogen, Waltham, MA, USA) and analyzed at a Leica TCS or Nikon spinning disk microscope.
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