Mouse anti cas9

Immunohistochemistry of wing imaginal discs was performed using standard procedures. Briefly, larva were dissected in ice cold PBS and fixed in 4% Paraformaldehyde in phosphate buffered saline (PBS) containing 0.05% Triton-X100 for 25 min at room temperature. Larva were washed three times in PBS containing 0.3% Triton-X100 (PBT) and then blocked for 1 hr at room temperature in PBT containing 1% heat-inactivated normal goat serum. Subsequently, larva were incubated with first antibody (mouse anti-Cas9 (Cell Signaling) 1:800; mouse anti-Cut (DSHB, Gary Rubin) 1:30; guinea pig anti-Sens (Boutros lab, unpublished) 1:300; rabbit anti-Evi [Port et al., 2008 (link)] 1:800) in PBT overnight at 4°C. The next day, samples were washed three times in PBT for 15 min and incubated for 2 hr at room temperature with secondary antibody (antibodies coupled to Alexa fluorophores, Invitrogen) diluted 1:600 in PBT containing Hoechst dye. Samples were washed three times 15 min in PBT and mounted in Vectashield (Vectorlabs). To visualize apoptotic cells wing discs expressing the apoptosis sensor GC3Ai (Schott et al., 2017 (link)) was fixed in 4% PFA, washed in PBT containing Hoechst and mounted in Vectashield.