Uranyl acetate

For transmission electron microscopy, extracellular vesicles were adhered to 100 mesh Formvar and carbon coated grids for 5 minutes at room temperature. Grids were washed once with water, stained with 1% uranyl acetate (Ladd Research Industries, Williston VT) for 1 minute, dried and viewed using a JEOL 1200 EXII transmission electron microscope. Images were captured using a Gatan Orius 600 digital camera (Gatan, Pleasanton, CA). Liver samples were collected from the CDAA-fed mice after a short liver perfusion with 10 mL of 4% paraformaldehyde in 0.15 M sodium cacodylate buffer, pH 7.4 by using a 21 G needle. Samples were immersed in modified Karnovsky’s fixative (2.5% glutaraldehyde and 2% paraformaldehyde in 0.15 M sodium cacodylate buffer, pH 7.4) for at least 4 hours, postfixed in 1% osmium tetroxide in 0.15 M cacodylate buffer for 1 hour and stained en bloc in 3% uranyl acetate for 1 hour. Samples were dehydrated in ethanol, embedded in Durcupan epoxy resin (Sigma-Aldrich), sectioned at 50 to 60 nm on a Leica UCT ultramicrotome, and picked up on Formvar and carbon-coated copper grids. Sections were stained with 3% uranyl acetate for 5 minutes and Sato’s lead stain for 1 minute. Grids were viewed using a JEOL 1200EX II (JEOL, Peabody, MA) transmission electron microscope and photographed using a Gatan digital camera (Gatan, Pleasanton, CA).

MPs were isolated from 3T3L1 or plasma described above and suspend in 1x PBS. For dynamic light scattering analysis, entire size was measured by Zetasizer nano ZS90 (Malvern). For Transmission electron microscope, MPs were adhered to 100 mesh Formvar and carbon coated grids for 5 minutes at room temperature. Grids were washed once with water, stained with 1% uranyl acetate (Ladd Research Industries, Williston VT) for 1 minute, dried and viewed using a JEOL 1200 EXII transmission electron microscope. Images were captured using a Gatan Orius 600 digital camera (Gatan, Pleasanton CA).

Formvar-carbon-coated copper grids (100 mesh, Electron Microscopy Sciences, Hatfield, PA) were placed on 20 μl drops of each sample solution displayed on a Parafilm sheet. After allowing material to adhere to the grids for 10 minutes, grids were washed 3 times by rinsing through 200 μl drops of milli-Q water before being left for 1 min on 2% (wt/vol) uranyl acetate (Ladd Research Industries, Williston, VT). Excess solution was removed with Whatman 3MM blotting paper, and grids were left to dry for a few minutes before viewing. Grids were examined using a JEOL JEM-1400Plus transmission electron microscope operating at 80 kV. Images were recorded using a Gatan OneView 4 K digital camera.