Transmission electron micrograph of Drosophila nephrocyte garland cell from ema mutant. ema mutant garland cells are filled with very large electron-lucent alpha and electron-dense alpha and beta vacuoles, many of which are 5–10 microns in diameter, whereas the largest vacuoles in wild type are ∼2 microns in diameter. In wild type, both types of vacuoles usually contain a single aggregate of electron-dense granular material that appears to be attached to the limiting membrane. However, the large vacuolar compartments in the mutant contain numerous electron-dense granular structures, suggesting they arise from homo-/heterotypic fusions of alpha and beta vacuoles. Samples for TEM 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. 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: 4000x. Image corresponds to Figure 2B, right ema panel in Kim et al. J Cell Biol. 188: 717-734. 2010. High magnification image taken from this micrograph is in CIL# 13469.
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