Samples were prepared for cryo-EM as above with 2 adaptations; crude fractions were not filtered before the 118 000 g step to allow the full range of native EVs to be included, and the final EV pellet was resuspended in 100 μL sterile filtered PBS. Three microliters of isolated EV sample was combined with 3 μL of 10 nm fiducials and placed onto glow-discharged, 300 mesh, lacey carbon grids and blotted for 2 to 3 seconds before being plunge frozen. Grids were visualized on a FEI Tecnai 20 200 kV LaB6TEM and images acquired using a FEI Ceta 4k x4k CCD detector.
Samples were immunogold labeled and prepared for transmission electron microscopy as follows; crude EV samples were diluted 1:16 and 3 μL of this sample was placed onto pioloform grids and the buffer removed with blotting paper. The grid was fixed in 4% paraformaldehyde, washed briefly in PBS, permeabilized in 0.05% saponin, blocked in 1% BSA and 0.01% saponin, incubated with anti-RFP (MBL International Corporation; PM005, 1:20), washed in PBS, incubated with anti-Rabbit 6 nm gold particles (Aurion; 806.011, 1:20), and washed in PBS. Grids were negatively stained in 0.3% uranyl acetate and 1.8% methyl cellulose (Sigma; M6385) on ice, air-dried and visualized on a FEI Tecnai 12 120 kV BioTwin Spirit Transmission electron microscopy and images acquired using a FEI Eagle 4k x4k CCD camera.
Samples were immunogold labeled and prepared for transmission electron microscopy as follows; crude EV samples were diluted 1:16 and 3 μL of this sample was placed onto pioloform grids and the buffer removed with blotting paper. The grid was fixed in 4% paraformaldehyde, washed briefly in PBS, permeabilized in 0.05% saponin, blocked in 1% BSA and 0.01% saponin, incubated with anti-RFP (MBL International Corporation; PM005, 1:20), washed in PBS, incubated with anti-Rabbit 6 nm gold particles (Aurion; 806.011, 1:20), and washed in PBS. Grids were negatively stained in 0.3% uranyl acetate and 1.8% methyl cellulose (Sigma; M6385) on ice, air-dried and visualized on a FEI Tecnai 12 120 kV BioTwin Spirit Transmission electron microscopy and images acquired using a FEI Eagle 4k x4k CCD camera.