Primary γh2ax antibody

For the γH2AX staining, cells were grown on a coverslip, were fixed with 3.7% formaldehyde. Permeabilized with 0.2% triton-X 100 for 5 mins, washed with cold PBS 3 times, and then blocking was done with 5% skimmed milk, 1hr at RT. Incubated with primary γH2AX antibody (Millipore) overnight at 4°C. Incubated with FITC conjugated anti-mouse secondary antibody (Jacksons) for 1hr in dark. After secondary incubation, the cells on coverslips were washed again with 0.1% PBST 3 times for 5 mins each and then mounted on glass slides with Prolong Antifade reagent (Invitrogen Molecular Probes, Eugene, OR, USA). Fluorescent images were captured with Nikon TE2000 inverted microscope at 60X objective (Nikon Instruments Melville, NY, USA). The number of foci/cell were counted and recorded for each cell individually. Foci in at least 60 cells were counted per biological replicate (BR), and the average number of foci/cell in each BR was further analyzed to determine the average number of foci/cell in 3 BR (13 (link), 24 (link)–26 (link)).

Sections (4 μm) of formalin-fixed, paraffin-embedded tissue were deparaffinized and antigen-retrieved using modified citrate buffer (Dako). Sections were incubated with primary γH2AX antibody (Millipore) at a dilution of 1/100 at 4°C for 3 hours. Secondary antibody (Alexa Fluor 488 Goat Anti-Mouse IgG, Invitrogen) was used at a dilution of 1/500 at room temperature for 1 hour. Sections were counter-stained with DAPI before imaging. For each section, images of 20 randomly selected image fields were acquired at a magnification of ×40 using ImagePro Plus software (Media Cybernetics). DAPI-stained nuclei were counted using ImagePro Plus, and nuclei containing more than 5 γH2AX foci were counted manually in a blinded fashion.