Fei tecnai g2 spirit

Samples were prepared for transmission electron microscopy (TEM) by the conventional negative staining procedure performed on. 5 different samples. In brief, 5 µL aliquots of EV suspension were sedimented for 2 min onto a 300 mesh, copper/carbon-coated grid and then negatively stained with 1% uranyl acetate and observed with a TEM Fei Tecnai G2 spirit at 80 Kv. For the immunogold procedure, fresh EVs were allowed to adhere to 300 mesh nickel formvar/carbon-coated grids, then exposed for 1h to the primary antibodies anti-CD9 (11-354-C100) and anti-CD63 (1B-343-C100), (both from ExBio, Prague, Czech Republic) and anti-flotilline (sc-74566, Santa Cruz Biotechnology, US), diluted 1:150 in PBS. The grids were then washed twice and finally incubated for 1h with the 10 nm gold-labelled secondary antibody (diluted 1:100 in PBS) (British Biocell, Cardiff, UK). After washing to eliminate any non-specific binding of secondary antibody, the samples were fixed using 1% glutaraldehyde, contrasted with 1% uranyl acetate, and observed with a TEM Fei Tecnai G2 spirit. The obtained digital images were processed with Image J software acquisition.

To identity EVs typical morphology a pool of 1.5 ml of concentrated control culture media was isolated using qEVs columns. Fractions 8 and 9 were collected. A total of 3 ml of isolated microvesicles were ultracentrifugated at 150.000 × g for 90 min at 4 °C. The EVs pellet was resuspended in 30 μl PBS 1X. The sample was fixed by 2% PFA. Negative staining was performed with 2% uranyl acetate. Photomicrographs were obtained using the transmission electron microscope FEI Tecnai G2 Spirit (FEI Europe, Eindhoven, Netherlands) using a digital camera Morada (Olympus Soft Image Solutions GmbH, Münster, Germany). EVs were identified under the microscope solely based on size and morphology.

For TEM analysis, sections were embedded in epoxy resin. First, samples were post-fixed with 1% osmium tetroxide (Electron Microscopy Sciences), 7% glucose in 0.1 M PB for 30 min at room temperature, washed in deionized water, and partially dehydrated in 70% ethanol. Afterward, the samples were contrasted in 2% uranyl acetate (Electron Microscopy Sciences) in 70% ethanol for 2 h at 4°C. The samples were further dehydrated and embedded in Durcupan ACM epoxy resin at room temperature overnight, and then at 70°C for 72 h. Once the resin was polymerized, immunolabeled sections were selected and cut into semithin (1.5 μm) and then into ultrathin sections (60–80 nm) using an Ultracut UC7 ultramicrotome (Leica). Ultrathin sections were placed on formvar-coated single-slot copper grids (Electron Microscopy Sciences) stained with lead citrate and examined at 80 kV on a FEI Tecnai G² Spirit (FEI Company, Hillsboro, OR) transmission electron microscope equipped with a Morada CCD digital camera (Olympus, Tokyo, Japan).

Vinyltrichlorosilan (VTCS) was obtained from abcr GmbH (Karlsruhe, Germany) and stored under nitrogen.Coverslips of size 26 × 76 mm were purchased from Menzel (Braunschweig, Germany) and para-aminophenol, para-nitrophenol and sodium borohydride were obtained from Sigma-Aldrich (St.Louis, MO, USA) and used as received. The holes in the glass slides were drilled using a KL450 ultrasonic drill (AQUARUS, Pappenheim, Germany). Sputtering targets (Mo, Pt, Ni, Cu, Au) were obtained from BALTIC Preparation e.K (Wetter, Germany). Sputtering was conducted with a CCU-010 HV coating unit (Safematic, Zizers, Switzerland). BLAUBRAND^® intraMARK (Brandt GMBH, Wertheim, Germany) 5 µL capillaries were purchased from the University of Zurich supply shop. An IPC high-precision multichannel dispenser (ISM930C) was used as peristaltic pump (Ismatec, Wertheim, Germany). Double distilled water was from a glass double distillery apparatus (GFL, Burgwedel, Germany) and was used to treat the slides prior to coating (SNFs, bagels); all other solutions were prepared from ultrapure water (Simplicity^® UV system) (Millipore, Billerica, MA, USA). SEM images were taken on a 450 Zeiss Gemini (Zeiss, Jena, Germany). TEM was performed using a FEI Tecnai G2 Spirit (FEI company, Hillsboro, OR, USA).