Immunofluorescence was performed as described28 (link),51 (link). Primary antibodies: γH2AX (1:400–500, MilliporeSigma, 05–636-I, clone JBW301), LBR (1:100, Abcam, ab32535, clone E398L). Secondary antibodies: Alexa Fluor, 488 (A11029), 568 (A11031) and 647 (A21236) (1:1000, Life Technologies). EdU was added 5 h before fixation.
Confocal images were collected using a Nikon Ti-E inverted microscope with a Yokogawa CSU-22 spinning disk head with the Borealis modification. Z-stacks were collected for 9 images at 0.4–0.6 μm spacing using a CoolSnap HQ2 CCD camera (Photometrics) and a 60x/1.40 NA Plan Apo oil immersion objective (Nikon) using Metamorph Software 7.10.2.240 (Molecular Devices). Alternatively, a Ti2 inverted microscope fitted with a CSU-W1 spinning disk system (Nikon) was used. Z-stacks were collected to cover the whole volume of cells at 0.4–0.6 μm spacing using a Zyla 4.2 sCMOS camera (Andor) and a 60x/1.40 NA Plan Apo λ oil objective and NIS-Elements 5.11.03 AR software (Nikon).
Confocal images were collected using a Nikon Ti-E inverted microscope with a Yokogawa CSU-22 spinning disk head with the Borealis modification. Z-stacks were collected for 9 images at 0.4–0.6 μm spacing using a CoolSnap HQ2 CCD camera (Photometrics) and a 60x/1.40 NA Plan Apo oil immersion objective (Nikon) using Metamorph Software 7.10.2.240 (Molecular Devices). Alternatively, a Ti2 inverted microscope fitted with a CSU-W1 spinning disk system (Nikon) was used. Z-stacks were collected to cover the whole volume of cells at 0.4–0.6 μm spacing using a Zyla 4.2 sCMOS camera (Andor) and a 60x/1.40 NA Plan Apo λ oil objective and NIS-Elements 5.11.03 AR software (Nikon).