Patm ser1981

Phospho-serine-139 H2A.X (γH2A.X) mouse monoclonal (Millipore JBW101) and rabbit monoclonal (Cell Signaling 20E3) are used at 1:500 dilution. Other antibodies are: 53BP1 (Cell Signaling 4937, 1:200), NBS1 (Novus Biologicals 100-143. 1:500), pATM Ser1981 (Millipore 05-740. 1:1000), and RPA-70 (Santa Cruz 28304. 1:200). Goat and donkey anti-mouse or anti-rabbit secondary antibodies conjugated to AlexaFluor 488, 568, or 647 are from Invitrogen and used at 1:500 dilution. Cells were grown on #1.5 glass coverslips (VWR 48366-227). Cells were fixed in 3.7% formaldehyde in PBS (pH 7.4) for 20 minutes at room temperature, washed at least 3 times in PBS, permeablized in 0.5% Triton X100 (Sigma) in PBS, washed at least 3 times in PBS, blocked in 4% BSA in PBS for 60 minutes at room temperature, incubated in primary antibody diluted in 4% BSA/PBS for 60 minutes at room temperature, washed at least 3 times in PBS, incubated in secondary antibody diluted in 4% BSA/TBS for 60 minutes at room temperature, washed at least 3 times in PBS, and counterstained with 1μM DAPI for 5 minutes at room temperature, washed in distilled water, and mounted in ProLong Gold or Prolong Diamond antifade reagent (Invitrogen) on glass microscope slides (VWR, 16004-422).