Gatan cryo transfer holder

EVs from approximately 30 brainstems were isolated as described above. Three μl of sample solution was applied to a glow-discharged Quantifoil grid (Quantifoil, R2/2) then loaded on a Leica EMGP plunger (Leica Biosystems). After blotting away excess liquid, the grid was quickly plunged into liquid propane. The cryo-grid was then transferred to a JEM-2100F electron microscope using a Gatan cryo-transfer holder (Gatan, Inc.). The electron microscope was operated at 200 KV with a field emission gun, and the sample was examined under minimum dose. The images were recorded with an OneView camera (Gatan, Inc.) at 50,000× magnification, corresponding to 2.16 Å/pixel resolution of specimen space. The “draw ellipse” function in ImageJ was used to measure vesicle diameters. The ellipse was fitted to the perimeter of each vesicle, whose diameter was taken as the average of the major and minor axes of the ellipse. A Mann-Whitney U-test was used to compare particle diameters from NTA and cryo-EM.

Quantifoil carbon R2/2 copper grids (Quantifoil Micro Tools GmbH, Jena, Germany) were glow discharged before sample freezing. The 3 µL of sample solution was applied to the Quantifoil grid and blotting was completed with a Leica EMGP plunger (Leica Microsystems Inc., Deerfield, IL, USA) at room temperature and 95% humidity. Blotting time was set to 3 s without waiting and draining time. The frozen hydrated grid was loaded on a pre-cooled Gatan cryo-transfer holder (Gatan, Inc., Pleasanton, CA, USA) and imaged under a JEOL JEM-2100F transmission microscope (JEOL USA, Peabody, MA, USA), operating at 200 kV. Images were taken at 30,000× magnification, corresponding to 0.27 nm pixel size at specimen space, and recorded on a Gatan OneView CCD (Gatan, Inc., Pleasanton, CA, USA) with SerialEM software (University of Colorado, Boulder, CO, USA) in low-dose mode. Images were processed using Fiji software [40 (link)]. Note: Cryo-EM parameters were optimized in order to capture high-resolution images. However, the background noise in micrographs of Ex Df + GFPAd and HMG Df + GFPAd was inevitable due to the high concentration of HSA in them.