Exosome Uptake and Ultrastructural Imaging

Apex2 staining was done afterward (Lam et al., 2015 (link)). Cells were grown in plastic six-well plates, incubated with 30 pM Flag-Apex2-emGFP-CD63 exosomes for 4 h and then fixed for 1 h on ice with 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer with 2 mM CaCl₂, pH 7.4. Cells were rinsed and treated for 5 min in 20 mM glycine on ice before the addition of 1.4 mM DAB and 0.03% H₂O₂ in cold buffer for 5–20 min. Cells were rinsed to stop DAB reaction and postfixed with 1% osmium tetroxide (Electron Microscopy Sciences) for 30 min in cold cacodylate buffer. Cells were rinsed, stained overnight at 4°C in 1% uranyl acetate in ddH₂O, dehydrated in a graded ethanol series, and flat-embedded in Embed-812 resin (Electron Microscopy Science). After polymerization at 60°C overnight, 50-nm sections were cut and poststained with lead citrate and uranyl acetate. Sections were imaged on an 80-kV Philips CM10 transmission electron microscope equipped with a Veleta camera (EMSIS).