Samples were stained and visualized essentially as described previously [10 (link), 90 (link)]. Aliquots of freshly thawed protein samples were adsorbed (10 μl) neat onto formvar-coated copper grids (Electron Microscopy Sciences, Hatfield, PA) for 1 min before 10 μl of 0.25% glutaraldehyde (ThermoFisher, Waltham, MA) was added and incubated for 1 min. Thereafter, grids were wicked dry using qualitative filter paper (VWR, Radnor, PA), washed twice with 10 μl MilliQ water and then stained with 1% uranyl acetate for 2 min. Grids were wicked dry as above and allowed to air dry for at least 10 min, stored at room temperature and then examined using a 1200EX microscope (JEOL).
For measurement of soluble protein aggregates, at least three grids per protein were mounted, and at least three images per protein were analyzed. Length, defined as the long axis of each fragment measured, was measured using FIJI [77 (link)] and binned into 8 nm segments [66 (link)] using the scale bar as a standard (2.58 pixels/nm). Particles were excluded from analysis if they were on the border of the image or did not have clearly defined edges (i.e. due to a clustering). All particles meeting these criteria within the image field of view were also analyzed for width, defined as the short axis of each measured fragment.
For measurement of soluble protein aggregates, at least three grids per protein were mounted, and at least three images per protein were analyzed. Length, defined as the long axis of each fragment measured, was measured using FIJI [77 (link)] and binned into 8 nm segments [66 (link)] using the scale bar as a standard (2.58 pixels/nm). Particles were excluded from analysis if they were on the border of the image or did not have clearly defined edges (i.e. due to a clustering). All particles meeting these criteria within the image field of view were also analyzed for width, defined as the short axis of each measured fragment.