Ema localizes to endosomes. Third instar larval garland cells expressing Ema-GFP fusion protein with endosomes labeled with a 5-min pulse of the fluid-phase endocytic tracer avidin-Cy3 followed by an additional 5-min chase. Ema localizes to functional endosomes. Third instar larvae were dissected in ice-cold PBS, equilibrated in the Schneider medium for 5 min at room temperature, pulsed with 10 µg/ml of Extravidin-Cy3 (Sigma-Aldrich) at room temperature for 5 min, rinsed thoroughly, and chased in Schneider medium. Samples were fixed in 4% PFA in PBS for 10 min at room temperature. 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 3A in Kim et al. J Cell Biol. 188: 717-734. 2010.