Jem 1400 plus electron

The isolated SEVs were prepared for transmission electron microscopy studies using negative staining. A 5 μL aliquot of SEV suspension was deposited onto 200 mesh carbon-coated EM grids. After washing, the samples were fixed for 5 min in 1% glutaraldehyde and then negatively stained with 2% aqueous solution of phosphotungstic acid. The grids were viewed in a JEM 1400 Plus electron microscope (JEOL Ltd, Tokyo, Japan) operating at 80 kV.

For electron microscopy, 3 μL aliquots (1–2 mg/mL) of the different samples were applied onto glow-discharged formvar/carbon-coated 200-mesh copper grids and incubated for 1 min. Grids were negatively stained with 1% (w/v) uranyl acetate (2 staining steps of 10 s) and air-dried for 5 min. Images of the fibrils were taken in a JEOL JEM 1400 Plus electron microscope. Images of type B* oligomers (α-syn_WT, α-syn_1-133, α-syn_1-122 and α-syn_1-110) were taken using a JEOL 1010 JEM electron microscope operated at 100 kV and equipped with a CCD camera (4 K × 4 K TemCam-F416, TVIPS). Images were recorded at a 65,000× nominal magnification with a pixel size of 15.50 μm (2.4 Å/px sampling rate). These images were processed following the Scipion2 processing workflow [64 (link)]. Images were CTF-corrected using CTFFIND4 [65 (link)]. Particles were automatically selected using Xmipp3 [66 (link)] and 2D-classified using Relion2 [67 (link)] and CryoSPARC (v2.14.2) [68 (link)]. The images shown in Figure 5 correspond to one of the larger and representative classes of each type B* oligomers obtained after 2D classification.