Carbon formvar coated copper grids

An in vitro polymerization reaction with 5C6 (3), RAL (7), LAS (13), and TAX (15) was conducted as described above. At the end of 30 min, the reaction was stopped by fixation in 0.5 % glutaraldehyde for 5 min. The microtubules were pelleted and resuspended in polymerization buffer without free tubulin. Resuspended samples were applied to glow-discharged 300 mesh carbon/formvar-coated copper grids (Electron Microscopy Sciences) and allowed to settle for 3 min. The grids were then washed by touching to water drops twice and stained with 1% uranyl acetate for 1 min. The grid was then dried and observed on the JEOL JEM-1400 TEM at 120 kV and images were taken using a Gatan Orius digital camera.

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