Filter paper

The EV separated from CTE and control brain tissue were analyzed by TEM. The EV sample (5µl) was adsorbed for 1 min to a carbon-coated mesh grid (# CF400-CU, Electron Microscopy Sciences) that had been made hydrophilic by a 20-s exposure to a glow discharge (25 mA). Excess liquid was removed with a filter paper (#1 Whatman). The grid was then floated briefly on a drop of water (to wash away phosphate or salt), blotted on a filter paper, and then stained with 0.75% uranyl formate (# 22451 Electron Microscopy Sciences) for 30 s. After removing the excess uranyl formate, the grids were examined, and random fields were photographed using a JEOL 1200EX TEM with an AMT 2k CCD camera.

Immediately following the measurement of ThT fluorescence intensity, pre-formed Aβ fibrils treated with individual hit compounds, or with DMSO as a control, were applied undiluted to Formvar/carbon-coated copper grids with 300 square mesh (Electron Microscopy Sciences) for 2 min. Grids were gently blotted on filter paper (Whatman) to remove excess fibrils, then washed twice in water and stained with 2% (w/v) uranyl acetate (Electron Microscopy Services) twice for 20 sec each, blotting on filter paper in between each step. Grids were air dried and imaged on a Tecnai G² Spirit BioTwin microscope (FEI) at 80 kV with a side-mount digital camera (AMT Imaging). TEM images were processed and analyzed using Fiji version 2.1.0/1.53c.