Indirect Immunofluorescence Microscopy Protocol

Indirect immunofluorescence was conducted as described previously [9 (link)]. Briefly, seeded cells were fixed, permeabilized, blocked, then incubated overnight at 4 °C with primary mouse antibodies: HA (H9658, Sigma-Aldrich, Oakville, ON, Canada, 1:1000) or γ-H2A.X (ab26350, abcam, UK 1:1000). The next day, cells were washed and then incubated for 1 h in the dark with secondary antibody mouse Alexa 647 (Invitrogen, Life Technologies, Burlington, ON, Canada, 1:1000). Coverslips were mounted with ProLong Gold containing 4′,6′-diamidino-2-phenylindole (DAPI) (Invitrogen, Life Technologies). Images were taken at 40× magnification with an Olympus BX51 microscope using Image-Pro Plus (v5.0) software (Media Cybernetics, Inc., Bethesda, MD, USA). All indirect immunofluorescence figures were generated using ImageJ (v2.1.0) and zoomed-in; single-cell panels were generated using the QuickFigures plug-in [29 (link)].