Tecnai 12 120 kv biotwin spirit tem

Samples were prepared for cryo-EM as above with two adaptations; crude fractions were not filtered prior to 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. 3 μl 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 2-3 seconds before being plunge frozen. Grids were visualised on a FEI Tecnai 20 200kV LaB6TEM and images acquired using a FEI Ceta 4k x4k CCD detector.
Samples were immunogold labelled and prepared for TEM 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, permeabilised 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 visualised on a FEI Tecnai 12 120kV BioTwin Spirit TEM and images acquired using a FEI Eagle 4k x4k CCD camera.