Transmission electron micrograph of Drosophila nephrocyte garland cell from wild type pulsed with the fluid-phase endocytic tracer HRP, detected by chromogenic staining (DAB). Most HRP-labeled endosomes in wild-type garland cells are about 100 nm in diameter. In the majority of the labeled endosomes, the HRP reaction product fills the luminal space. Third instar larvae were dissected in ice-cold PBS, equilibrated in HL-3 buffer at room temperature, incubated in 0.7% HRP (Sigma-Aldrich grade VI) for 5 min at room temperature, rinsed thoroughly, and chased for 10 min at room temperature. Samples were fixed for 1 h at 4°C in 2% paraformaldehyde, 2% glutaraldehyde, and 1% tannic acid in 0.1 M cacodylic acid buffer, pH 7.2, and processed for DAB reaction as described previously (Kosaka and Ikeda, 1983). Garland cells were dissected out and fixed for 1 h at 4°C in 2% paraformaldehyde, 2% glutaraldehyde, and 1% tannic acid in 0.1 M cacodylic acid buffer, pH 7.2. Then, the samples were postfixed in 1% OsO4 in 0.1 M cacodylic acid buffer, pH 7.2, for 1 h at room temperature, stained en bloc with 1% uranyl acetate, dehydrated in a grade series of ethanol and propylene oxide, and embedded in epon resin (Electron Microscopy Sciences). Blocks were sectioned in an ultramicrotome (RMC Products) at ∼70-nm thickness with a Delaware Diamond knife and post-stained for 1 h in Reynolds lead citrate and uranyl acetate. Electron micrographs were taken on a transmission electron microscope (H-7500; Hitachi) using an AMT camera system. Mag: 40000x. Image corresponds to Figure 4C, top panel in Kim et al. J Cell Biol. 188: 717-734. 2010.