Ema localizes to endosomes. Third instar larval garland cells expressing Ema-GFP fusion protein and stained for the multivesicular body ESCRT protein Hrs (red). Ema does not localize to MVB. Wandering third instar larvae were dissected in ice-cold PBS, and fixed with Bouin’s solution for 5 min. Blocking and antibody incubation were performed in PBS containing 0.1% Triton X-100. Anti-HRS-FL (Lloyd et al., 2002) was used at 1:1,000. Cy3-conjugated secondary antibody (Jackson Immunoresearch Laboratories, Inc.) at 1:1,000. Confocal images were acquired with a confocal microscope (model C1; Nikon) and accompanying EZ-C1 software using argon (excitation at 488 nm) and HeNe (excitation at 543 and 633 nm) lasers and a 60x Plan-Apochromat NA 1.4 objective (Nikon) at room temperature. Samples for each experiment were processed using the same confocal gain setting. Image corresponds to Fig 3D in Kim et al. J Cell Biol. 188: 717-734. 2010.