Mega view 2

hBMECs were cultured on poly-l-lysine-coated coverslips overnight prior to infection by tachyzoites of N. caninum at a host:parasite ratio of 1:2 (i.e., MOI; multiplicity of infection of 2). Twenty-four hours after incubation, cells were fixed in 1% glutardialdehyde-4% formaldehyde in 0.1M PBS at 4 °C for at least 2 h before processing. Samples were washed in 0.1 M PBS at 4 °C for 12 h, followed by a secondary fixation in 1% osmium tetraoxide-1.5% K₄Fe(CN)₆ in 0.1 M PBS (pH 7.2). Samples were washed twice in distilled water for 30 min and dehydrated in ethanol 50, 50, 70, 70, 90, 90, and 96% for 10 min and twice in ethanol 100% for 15 min. Samples were impregnated in equal volumes of epoxy resin (LX112) and ethanol 100% for 60 min at ambient temperature, followed by pure Epoxy resin (LX112) for 60 min at 37 °C. Resin was allowed to polymerize for 12 h at 60 °C. Sectioning of blocks was performed on a type LKB IV ultramicrotome at 40 nm, and then the sections were collected on a copper 200-mesh grid. Sections were incubated with 6% uranyl acetate for 10 min, followed by lead citrate for 1 min, before they were viewed under a Philips Morgangi 268 transmission electron microscope connected to a charge-coupled device camera (MegaView II).

To detect the internalization of ASC-exosomes by the cells, exosomes labelled with USPIO nanoparticles, that allowed the visualization by TEM, were used. The NSC-34(G93A) cells were seeded at a density of 2 × 10⁴ with the presence of exosomes-USPIO (0.2 µg/mL, corresponding to 6–8 × 10⁵ particles/mL) in the culture medium for 6 h. After the incubation time, the cells were washed with PBS, trypsinized and centrifuged. For ultrastructural morphology of cells, the pellet was fixed in 2% glutaraldehyde in Sorensen buffer (pH 7.4) for 2 h. The samples were post-fixed in 1% osmium tetroxide (OsO₄) for 2 h, cut, dehydrated in graded concentrations of acetone and embedded in Epon-Araldite mixture (Electron Microscopy sciences, Fort Washington, PA, USA). The semithin sections (1 µm in thickness) were examined by light microscopy (Olympus BX51, Olympus Optical, Hamburg, Germany) and stained with toluidine blue. The ultrathin sections were cut at a 70 nm thickness, placed on Cu/Rh grids with Ultracut E (Reichert, Wien, Austria). TEM images were acquired with a Philips Morgagni TEM operating at 80kV and equipped with a Megaview II camera for digital image acquisition.