Em 109

TEM analyses of NPs were performed by means of a transmission electron microscope (Zeiss EM109), prepared by slow evaporation of one drop of aqueous solution of the NPs placed on a formvar/carbon-coated copper mesh grid and air-dried. For ex vivo analyses, small portions of MCF-7 tumour samples were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, for 2 h. After one rinsing with phosphate buffer, specimens were post-fixed in 1.5% osmium tetroxide for 2 h, dehydrated by 70, 90 and 100% ethanol, and embedded in epoxy resin (PolyBed 812 Polysciences Inc., USA). Ultrathin sections were examined by the Zeiss EM109 microscope.

Exosomes were fixed in 2% paraformaldehyde (PFA) solution and applied on Formvar-carbon coated grids (TAAB Laboratories). Samples were washed with PBS and fixed for 5 min with 1% glutaraldehyde. Fixated grids were washed with deionized water and subsequently contrasted for 5 min with an uranyl-oxalate solution (4% uranyl acetate, 0.15 M oxalic acid, pH 7, Sigma-Aldrich). At the end, grids were air dried for 10 min and examined under a Zeiss EM 109 with an accelerating voltage of 80 kV. For immunogold staining, 5 μl of resuspended paraformaldehyde fixed exosomes were transferred on glow discharged Formvar-carbon coated nickel grids. After washing with PBS, the grids were incubated for 3 min with 50 mM glycine/PBS. The specimens on the grids were permeabilized with 0.1% saponin for membrane protein labeling before they were washed and blocked with 0.5% BSA. Exosomes were incubated with the following monoclonal primary antibodies: mouse anti CD9 (clone HI9a, antibodies-online, 1:10), mouse anti CD81 (sc-166,029, Santa Cruz, 1:20) and mouse anti CD90 (clone 5E10, BD, 1:40), washed and labeled with a goat anti mouse secondary antibody conjugated to 10 nm gold particles (EM.GMHL10, BBI Solutions, 1:15). Grids were contrasted as described above. Observations were carried out with a Zeiss EM 109 with an accelerating voltage of 80 kV (Zeiss Oberkochen, Germany).

A single Pterobdella specimen for TEM microscopy was initially preserved in 3% glutaraldehyde buffered with 0.1 M phosphate and 0.3 M sucrose (pH 7.8). Samples were washed in 0.1 M sodium cacodylate with 24% sucrose and post-fixed with 1% OsO4 in 0.1 M sodium cacodylate for 1 h. Samples were stained in 3% uranyl acetate in 0.1 M sodium acetate buffer for 1 h, dehydrated in ethanol and embedded in Spurr’s resin. Thick sections (0.4 μm) were stained with methylene blue and examined using a Nikon E80i microscope, while thin sections (70 nm) were stained with lead citrate and examined using a Zeiss EM 109 transmission electron microscope.