Gold conjugated goat anti mouse immunoglobulin g igg serum

For electron microscopy, purified Exo and MV from 0.1 ml of plasma were processed for scanning electron microscopy (SEM) and transmission electron microscopy (TEM) as previously described (Shively and Miller, 2009 (link); Paolino et al., 2021b (link)).
For the immunoelectron microscopy, the samples on the grids were incubated with anti-CD81 monoclonal antibody (mAb B11, Santa Cruz Biotechnology, Heidelberg, Germany), and with 10 nm gold-conjugated goat anti-mouse immunoglobulin G (IgG) serum (Sigma-Aldrich, St. Louis, MO). Finally, nanovesicles were observed with a PHILIPS EM208S transmission electron microscope (FEI-ThermoFisher) (Federici et al., 2020 (link)).

Either purified exosomes and microvesicles from healthy donor NK cells were suspended in PBS and adsorbed on carbon-coated grids for electron microscopy, accordingly to Thery group's protocol (28 (link)) with slight modifications. Samples were air dried, and all the successive passages were carried out by floating the grids on drops of different solutions. First, the grids were floated on anti-CD81 (mAb B11, Santa Cruz Biotechnology, Heidelberg, Germany) or anti-CD56 (mAb 123C3, Santa Cruz) monoclonal antibodies, rinsed on buffer and incubated on 10 nm gold-conjugated goat antimouse immunoglobulin G (IgG) serum (Sigma-Aldrich, St. Louis, MO). Then, samples were rinsed and incubated on 2% paraformaldehyde. Finally, after rinsing on water, the samples were contrasted with 2% ammonium molybdate (“positive-negative” contrast) and dried with a filter paper. Samples were observed with a PHILIPS EM208S transmission electron microscope (FEI-Thermo Fisher).