Ga508

Cells were fixed in 4% paraformaldehyde for 15 min, washed with PBS (3 × 5min), and blocked in the presence of 0.1% Triton X-100 and 2% BSA for 1h at room temp. Cells were then incubated in primary antibody overnight, washed with PBS (3 × 5min), incubated with the secondary antibody in 2% BSA for 1hr, washed (4 × 10mins, adding 1mg/ml DAPI at the second wash), mounted using Vectashield (Vector Labs) and imaged using a Leica TCS-SPE confocal microscope at 10x,40x or 63x. Antibodies use in immunostaining were: TBX18 (Santa Cruz sc-514486, 1:200), Oct 4 (Novus Biologicals, NB11–1:150), p16 (Invitrogen MA5–14260, 1:250), p21 (Cell Signaling 2947S, 1:200), GATA3 (Cell signaling D13C9, 1:250), CGB (DAKO GA508; 1:200). Slides were rinsed with PBS-T and incubated with secondary antibodies: DAPI (0.02 µg/mL, Molecular Probes), goat anti-rabbit 488, and goat anti-mouse 647 (1:500, Molecular Probes) for 2 h at room temperature in blocking buffer. (EBdCas9 or NCdCas9 were visualized using endogenous mCherry expression) (SI Table S6).