The endosomal compartment is enlarged in the ema mutant. Colocalization between DVGLUT (red) aggregates and the endosomal ESCRT protein Hrs (green, also known as Vps27) in motoneuron cell bodies of the ventral nerve cord of the ema mutant. 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-DVGLUT (Daniels et al., 2004) antibody was used at 1:10,000; anti-HRS-FL (Lloyd et al., 2002) was used at 1:1000. Secondary antibodies (Alexa 488-/Cy3-conjugated) were used at 1:1000. After staining, specimens were equilibrated in 70% glycerol in PBS and mounted with VectaShield (Vector Laboratories). 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 Figure 2A, bottom 3 panels in Kim et al. J Cell Biol. 188: 717-734. 2010.
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