Ultrathin Cryosectioning and Immunogold Labeling for Exosome Visualization

For ultrathin cryosectioning and immunogold labelling, the cells were fixed with 2% PFA or with a mixture of 2% PFA and 0.2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Cells were processed for ultracryomicrotomy (70 nm of thickness), immunolabelled with anti-CD63 and immunogold-labelled using PAG10 as reported38 (link). For EM of the isolated exosomes, a drop of exosomes suspended in PBS was deposited on Formvar-carbon-coated electron microscopy grids, fixed as above, immunolabelled and stained using the method described for ultrathin cryosections38 (link). All samples were analysed using a FEI CM120 electron microscope (FEI Company), and digital acquisitions were made with a numeric camera (Keen View; Soft Imaging System, SIS, Germany).