Anti phospho histone h2ax γh2ax

Cells on coverslips were plated on ice for 0.5–1 min before pre-extracted by ice-cold PBS+0.5% Triton for 5 min. Then, cells were fixed by 3% paraformaldehyde/2% sucrose for 10 minutes at RT. Cells were washed twice with PBS-T (0.01% Tween) and incubated with primary antibodies (RPA70/RPA1 Ab, Cell Signaling Technology #2267; anti-phospho-Histone-H2A.X/γ-H2AX, Millipore 05-636 clone JBW301; anti-PARP1, Abcam ab227244; anti-XRCC1, Abcam ab134056; anti-53BP1, Novus Biological, NB100-304) in filtered DMEM + 10% FBS (alternatively add 3% BSA) at 37°C for 1h. After 3x PBS-T washing, coverslips were incubated with appropriate secondary antibodies (in DMEM + 10% FBS) and DAPI. Finally, after washing with PBS-T (x3), coverslips were mounted with Prolong (Invitrogen, P36930). For all assays above, images were collected by fluorescence microscopy (Axioplan 2 imaging and Axio Observer, Zeiss) at a constant exposure time in each experiment. Representative images were processed by ImageJ software. Mean intensity of immunofluorescence for each nucleus were measured with Cell Profiler software version 3.1.5 from Broad Institute.