Drosophila Larval Dissection and Immunostaining

The genotype of the animal shown in Fig. 8 was y w hsFlp/Y; ex⁶⁹⁷FRT42D ubi-GFP/FRT42D, hpo42 (link)43 (link)44 (link)45 (link)46 (link)47 (link); hh-Gal4/msn-RNAi. The msn-RNAi line was the verified TRiP line BSC2879146 (link). Wandering third instar larvae of this genotype were dissected in cold 1 × phosphate-buffered saline (PBS; pH 7.2) and fixed in 4% formaldehyde in 1 × PBS for 50 min. Samples were permeabilized in 1 × PBT (1 × PBS+0.3% Triton X-100) and blocked in 5% normal donkey serum in PBT. Primary antibodies were incubated overnight at 4 °C: mouse anti-β-galactosidase (Promega, 1:2,000) and rat anti-Cubitus interruptus (DSHB, 1:150). Secondary antibodies were incubated for 3 h at room temperature: donkey anti-mouse Cy3 (Jackson, 1:600) and donkey anti-rat Cy5 (Jackson, 1:600). Samples were mounted in Vectashield (Vector Labs) and imaged using an Olympus FV1000. Images were processed using ImageJ, and figures prepared using Adobe software.