Immunofluorescent detection of Cas9 in Hela cells transiently transfected with a Flag-tagged Cas9 expression vector using a Cas9 monoclonal antibody (Product # MA5-23519). 48 hours post transfection, the cells were fixed in 3.7% formaldehyde, permeabilized in 0.5% Triton-X-100 and blocked in PBS containing 2% BSA for 2 hours at RT. The cells were stained with the Cas9 (fig 4A and B) or with an anti-Flag (fig 4C) antibody at 4°C overnight, followed by incubation with an anti-mouse secondary antibody coupled to AlexaFluor 488 for 1 hr at RT. Nuclei were counter-stained with Hoechst 33342 (bottom).