400 mesh carbon coated copper grid

Negative stain electron microscopy was used to examine the ultrastructural characteristics of S, NS, and PMCA fibrils. Aliquots (5 μL of 69 μM α-syn fibril preparations) containing recombinant α-syn fibrils were loaded on to freshly glow discharged 400 mesh carbon-coated copper grids (Electron Microscopy Sciences) and adsorbed for 1 minute. Grids were then washed with 50 μL each of 0.1 M and 0.01 M ammonium acetate and stained with 2x50 μL of freshly filtered 2% uranyl acetate. Once dry, grids were visualized with a Technai G20 transmission electron microscope (Thermo Fisher FEI) using an acceleration voltage of 200kV. Electron micrographs were recorded with an Eagle 4kx4k CCD camera (Thermo Fisher FEI). Fibril lengths were measured for a total of 62 S fibrils and 47 NS fibrils stemming from three independent fibril preparations. No overlapping or otherwise obscured fibrils were included in the length measurements. Measurements were determined in Adobe Photoshop software using the “Ruler” tool, which allows single pixel accuracy in the measurements with ~0.5 nm / pixel at the image magnification that was used. Mean fibril lengths in three independent preparations were compared using a two-tailed, unpaired t-test.