Tecnai t10 electron microscope

A total of 5 μg of RBC‐EVs was diluted in PBS and placed on 200‐mesh carbon‐coated copper grids at RT for 2 min. The excess suspension was removed using filter paper. Then, the RBC‐EVs were negatively stained with uranyl acetate at RT for 5 min, washed twice with PBS, dried and examined under an FEI Tecnai T10 electron microscope (FEI, Hillsboro, OR, USA) operating at 100 kV.

Full methods used have been published previously^{9 (link)}. Briefly samples were loaded onto carbon-coated 300 mesh copper grids (Electron microscopy Sciences) that were glow discharged for 40 seconds using an PELCO easiGLOW™ glow discharge unit (Ted Pella Inc, USA). Samples were left to bind for 2 minutes, blotted, washed briefly in one water drop, blotted, and then stained with either NanoW stain (Nanoprobes) 2 × 1 min or 2% (w/v) uranyl acetate in water for 45 sec, then blotted and air-dried. Grids were inserted into the microscope using a dedicated sample holder for mouse prions with strict adherence to risk assessment and microbiological containment level 2 safe working practice. The sample holder was decontaminated directly after use by exposing to plasma using a Fischione 1020 plasma cleaner. Images were acquired on an FEI Tecnai T10 electron microscope (FEI, Eindhoven, NL) operating at 100 kV and recorded on a 1 k × 1 k charged couple device (CCD) camera (Gatan) at a nominal magnification of 44,000 with a pixel size of 3.96 Å. Fibril dimensions were measured in ImageJ^{41 (link)} and IMOD^{42 (link)}. Because of their helical twist, prion rods and recombinant PrP fibrils viewed on a surface alternate between wider, face-on and narrower, edge-on views of the structure. The dimensions reported here were measured on the widest parts of the fibrils.