Fei cm120 electron microscope

For ultrathin cryosectioning, melanocytes 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) and stained using the method described for ultrathin cryosections^{47 (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).

For ultrathin cryosectioning and immunogold labeling, control and siRNA inactivated cells were allowed to internalize cell surface-bound EGF-Alexa (4 µm/ml) for 7 min. Cells were washed at 4°C, then fixed with a mixture of 2% PFA and 0.2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Cells were processed for ultracryomicrotomy and single or double immunogold labeled with the indicated antibodies detected using Protein A conjugated to gold (PAG10 or PAG15; purchased from Cell Microscopy Center, Utrecht, The Netherlands) as reported [52] (link). For HRP cytochemistry and conventional Electron Microscopy, control and inactivated HeLa cells grown on coverslips were grown in serum free media and allowed to internalize Horse Radish Peroxidase (HRP) (10 mg/ml, Sigma, Germany) for 7 min at 37°C. After washing at 4°C, cells were fixed with a mixture of PFA 2% and Glutaraldeyde 1.5%. Cells were washed with Cacodylate buffer 0.2M and further rinsed in Tris-HCl pH 7.6 before incubation with di-aminobenzidine and H2O2 for 20 min. Cells were further fixed with 2.5% glutaraldehyde in 0.1M cacodylate buffer for 24 h and processed as described previously [53] (link). All samples were analyzed using a FEI CM120 electron microscope (FEI Company) at 80 kV, and digital acquisitions were made with a numeric camera (Keen View; Soft Imaging System, SIS, Germany).

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).