Ultracut uct

WT and SOCS3-null AS-M.5 cells were seeded at a density of 1 × 10⁶ cells per ml into 6-well plates and onto Thermanox coverslips (13 mm diameter) for culturing to confluency. The cells were then fixed in 1.5% (w/v) glutaraldehyde in 0.1 M sodium cacodylate buffer at 4 °C for 1 h. Following three washes in 0.1 M sodium cacodylate buffer in 2% (w/v) sucrose, the cells were incubated with 1% (w/v) osmium tetroxide/0.1 M sodium cacodylate for 1 h, washed three times in distilled water, and incubated in 0.5% (w/v) uranyl acetate in the dark for 1 h. Following two rinses in distilled water, cells were dehydrated in stepwise alcohol increments (30–100% (v/v)) and incubated overnight in a 1:1 mix of propylene oxide/TAAB araldite Epon 812 resin. The propylene oxide was then allowed to evaporate to leave pure resin, which was changed twice before the sample was embedded in fresh resin which was allowed to polymerise at 60 °C for 48 h. Ultrathin sections were cut using a Leica Ultracut UCT and a Diatome diamond knife, contrast stained with aqueous 2% (w/v) methanolic uranyl acetate and Reynolds lead citrate, and viewed using a LEO 912AB TEM (Carl Zeiss) at an accelerating voltage of 120 kV. TIF images were captured using an Olympus Soft Imaging System and image contrast modified using Adobe Photoshop CS.

Cells collected 4 h after TACE for transmission electron microscopy (TEM) analysis were detached with scraper, washed in PBS, and then fixed in 4% paraformaldehyde solution for 48 h. Samples were treated with 1% osmium tetroxide (OsO4) for 1 h at 4 °C before being dehydrated in increasing grades of alcohol (10 min each: 50%, 70%, 90%; 2 × 5 min: 100%) and embedded in an epoxy resin. Ultrathin sections (70–100 nm) were cut with a Leica Ultracut UCT equipped with a diamond knife (Diatome), transferred to copper grids, stained with 2% aqueous uranyl acetate, and examined with a H-7650 electron microscope at 80 KV. The purified EVs were left on formvar/carbon copper grids, and droplets of EVs were cleared with filter papers. The grids were negatively stained with 2% uranyl acetate for 3 min. After washing with PBS, images were obtained by TEM at 80 KV (H-7650; Hitachi). For EV morphology analysis, 10 μl of EV aliquot sample was applied to a glow-discharged 300-mesh R2.0/2.0 Quantifoil grid. The specimen grid was blotted by Whatman #1 filtration paper and then immediately plunged into liquid ethane to rapidly form a thin layer of amorphous ice by using a Thermo Scientific Vitrobot Mark IV system. The grid was transferred under liquid nitrogen to a Thermo Scientific Glacios Cryo-TEM. Images were recorded by a Thermo Scientific Falcon direct electron detector.