Ultracut em uc7 ultramicrotome

The ECIV isolate was propagated in a 75 cm² flask of EPC cells until CPE was observed. The supernatant from the infected flask was discarded, and the monolayer was fixed in 15 mL of modified Karnovsky’s fixative (2P + 2G, 2% formaldehyde prepared from paraformaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer pH 7.4) at room temperature for 1 h. The monolayer was then washed in cacodylate buffer, scraped off the flask, and pelleted. The pellet was shipped via PBS overnight on ice packs to the University of Texas Medical Branch Department of Pathology Electron Microscopy Laboratory (UTMB-EML). At UTMB-EML, the cell pellet was washed in cacodylate buffer and left in 2P + 2G fixative overnight at 4 °C. The next day, the cell pellet was washed twice in cacodylate buffer, post-fixed in 1% OsO₄ in 0.1 M cacodylate buffer pH 7.4, en bloc stained with 2% aqueous uranyl acetate, dehydrated in ascending concentrations of ethanol, processed through propylene oxide, and embedded in Poly/Bed 812 epoxy plastic (Polysciences, Warrington, PA, USA). Ultrathin sections were cut on a Leica ULTRACUT EM UC7 ultramicrotome (Leica Microsystems, Buffalo Grove, IL, USA), stained with 0.4% lead citrate, and examined in a JEM-1400 electron microscope (JEOL USA) at 80 kV.

EVs were resuspended in 2% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) and loaded on carbon Formvar-coated copper grids (Electron Microscopy Sciences, Hatfield, PA, USA), which were subsequently stained with uranyl acetate (Electron Microscopy Sciences, Hatfield, PA, USA). In the case of cells, they were fixed overnight in 2% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) in 0.1 M phosphate buffer (Electron Microscopy Sciences, Hatfield, PA, USA), post-fixed for 1 h in 2% osmium tetroxide (Electron Microscopy Sciences, Hatfield, PA, USA) in 0.1 M phosphate buffer, dehydrated through a series of graded ethanols (Pharmco-Aaper, Brookfield, CT, USA), and embedded in EM-bed (Electron Microscopy Sciences, Hatfield, PA, USA). The glass coverslip was dissolved in hydrofluoric acid (Sigma-Aldrich, St. Louis, MI, USA). Then, 100 nm sections were cut on a Leica Ultracut EM UC7 ultramicrotome (Leica Microsystems, Buffalo Grove, IL, USA) and stained with uranyl acetate and lead citrate (Electron Microscopy Sciences, Hatfield, PA, USA). The grids were viewed at 80 kV in a JEOL JEM-1400 transmission electron microscope (JEOL, Peabody, MA, USA) and images captured by an AMT BioSprint 12 (AMT Imaging Systems, Woburn, MA, USA) digital camera

CM61 CTCs were grown in either low attachment or adherent conditions. Adherent cells were grown on cover-slips while suspension cells were pelleted and fixed. They were both fixed overnight in 2% glutaraldehyde in 0.1 M phosphate buffer, post-fixed for 1 h in 2% osmium tetroxide in 0.1 M phosphate buffer, dehydrated through a series of graded ethanols, and embedded in EM-bed (Electron Microscopy Sciences, Fort Washington, PA, USA). The glass cover-slip was dissolved in hydrofluoric acid. Then, 100 nm sections were cut on a Leica Ultracut EM UC7 ultramicrotome (Wetzlar, Germany) and stained with uranyl acetate and lead citrate. The grids were viewed at 80 kV in a JEOL JEM-1400 transmission electron microscope (Tokyo, Japan) and images were captured by an AMT BioSprint 12 digital camera (Woburn, MA, USA).