Immunofluorescent staining of HT1080 cells (ATCC CCL-121). Cells were seeded in a 96 well imaging plate at ~10,000 cells per well. After overnight incubation, cells were either mock treated (PBS, left) or exposed to hydrogen peroxide (400uM, right) for 30 minutes and allowed to recover in media for 30 minutes. After treatment cells were stained using the alcohol perm protocol and the anti-53BP1 antibody. The second step reagent was Alexa Fluor® 488 goat anti-mouse IgG (Invitrogen). The image is a confocal collapsed stack, taken on a BD Pathway™ 855 bioimaging system with a 40x objective. This antibody also stains A549 (ATCC CCL-158), HeLa (ATCC CCL-2) and U-2 OS (ATCC HTB-96) cells and can be used with either fix/perm protocol.