Anti 53bp1 antibody

H460 and A549 cells were grown as monolayers on chamber slides with plastic bottom (Nunc Lab-Tek, Roskilde, Denmark) and were treated with DMSO or ganetespib (25 nM, 50 nM) 24 hours after seeding into culture chambers. Five hours after ganetespib treatment, the cells were subjected to irradiation at dose of 2 Gy. Thirty minutes, 4 hours, 24 hours and 48 hours after irradiation, cells were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature and washed in PBS. The cells were then permeabilized in 0.5% Triton X-100 for 10 min, and blocked in PBS with 3% BSA (Bovine serum albumin) for 20 min at room temperature. The cells were sequentially incubated with anti-53BP1 antibody (Cell Signaling Technology, Danvers, MA, USA) at 1:100 dilution overnight at 4 °C and Alexa Fluor® 488 Conjugate secondary antibody (Cell Signaling Technology, Danvers, MA, USA) at a 1:1000 dilution for 1 hour at 37°C in PBS with 1.5% BSA and washed three times in PBS. Nuclei were counterstained with 1:500 4-diamidino-2-phenylindole dihydrochloride (DAPI) in PBS. Cover glasses were mounted in Vectashield (Vector Laboratories). Fluorescence images were captured with Leica fluorescence microscope equipped with a CCD camera and images were imported into Advanced Spot Image analysis software. DNA repair foci were quantified using ImageJ software (NIH public domain).