Immunofluorescent Staining of γH2AX Foci

The immunofluorescent staining of cell nuclei and γH2AX foci was performed according to the following protocol; cells grown on cover slips were washed in PBS and fixed in 4 % paraformaldehyde (in 1xPBS, pH 7.4) for 15 min at room temperature. After three washing steps with PBS, cells were permeabilized using 0.1 % Triton-X 100 (in 1xPBS, pH 7.4) for 15 min at room temperature and then incubated with the blocking reagent (5 % Bovine serum albumin in 1xPBS, pH 7.4) for 45 min. The primary antibody anti-γH2AX (Ab26350, Abcam) was diluted to 1:1000 in 1 % Bovine serum albumin, (in1xPBS pH 7.4) and added to the cells for 2 h at room temperature. After the incubation, cells on cover slips were washed three times in PBS and the fluorescent-labelled secondary antibody diluted 1:500 in the same buffer was added to cells (IgG-Alexa488, Cell Signaling #4408). The samples were stored in the dark at room temperature for 1 h. After washing, the DNA was stained with 49-6-diamidine-2-phenyl indole (DAPI, Invitrogen) diluted to a final concentration 1 μg/ml in the same buffer for 5 min at room temperature. Cells were then washed in PBS and mounted with the anti-fade medium (Vectashield).
Then cells were imaged using a confocal fluorescent laser scanning microscope (FluoView1000, Olympus) with a 60 × oil objective with numerical aperture (N.A.) equal to 1.35. In addition, cells were visualized using a conventional wide-field fluorescent microscope (Keyence BZ-8100E) with a 20× objective with N.A. equal to 0.4. As a result, TIF-images were obtained, where nuclei and foci were detected by blue and green channels, respectively.