Phospho h2a x s139

For immunofluorescence, 15,000 HT1080, HT1080-BRCA1^KO, or HT1080-PALB2^KO cells were plated on coverslips 24 hours prior to treatment. Cells were either irradiated with 8Gy of ionizing radiation or left unirradiated. Cells were allowed to recover for 2 hours, fixed with 10% formalin for 15 mins, washed with 1x PBS, permeabilized with 0.5% Triton-X and 0.2M HCl for 10 minutes, washed again with 1x PBS and blocked with a 4:1 ratio of 10% BSA in PBS-Tween 0.05% and 20% FBS in PBS for 75 minutes. After blocking, cells were rinsed with PBS and incubated with primary antibodies against Rad51 (Mouse Anti-Rad51 monoclonal, Abcam ab213, diluted at 1:500) or γH2AX (Rabbit mAb Phospho-H2A.X S139, Cell Signaling #9718, diluted at 1:1000) overnight at 4°C. The following day the cells were washed with PBS and incubated with secondary AlexaFluors (Goat anti-Mouse IgG (H+L) Secondary Antibody AlexaFluor 568; Goat anti-Rabbit IgG (H+L) Secondary Antibody AlexaFluor 488, diluted at 1:500) in blocking buffer for 1 hour. Cells were again rinsed with PBS and subsequently dH2O. Coverslips were gently dried and fixed onto slides with ProLongGold + DAPI dye and imaged on an ECHO Revolve Light Microscope. Images were compiled and analyzed using ImageJ.