Mouse anti histone h2ax pser139

HeLa Kyoto cells or Flp-In T-REx 293 cells were fixed and processed for immunofluorescence as described45 (link). In brief, cells were fixed in 4% paraformaldehyde for 10 min at the room temperature followed by ice-cold methanol for 1 min. The cells were then wash with 1 × phosphate-buffered saline (PBS) and permeabilized with 0.5% Triton X-100 for 5 min before antibody staining. The primary antibodies used were as follows: mouse anti-histone H2AX pSer139 (1:1,000, Millipore 05-636-1), mouse anti-α-tubulin (1:5,000, Sigma-Aldrich T9026), rabbit anti-GFP (1:5,000, Abcam ab290), mouse anti-cyclin A (1:200, Santa Cruz sc-56299), mouse anti-FLAG (1:1,000, Sigma-Aldrich A8592). Secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 555 (1:1,000, Molecular Probes) were used for detection. DNA was stained with 4,6-diamidino-2-phenylindole. Images were acquired using Zeiss AXIO Imager M1 with a × 40 EC-Plan-Neofluor lens and Hamamatsu photonics camera under the control of Volocity software. Images were processed using Adobe Photoshop.