Transmission electron microscopy

Five microliters of TDP-43 proteins or Aβ samples from ThT assay were deposited onto 400-mesh Formvar carbon-coated copper grids (EMS electron Microscopy Sciences, Hatfield, PA, USA) for 1 min. The grids were blotted, washed with droplets of Milli-Q water, and stained with 2% uranyl acetate. The samples were examined with a FEI Tecnai G2 F20 Super TWIN transmission electron microscope with an accelerating voltage of 120 kV. For immunogold labeling, 5 µl Aβ fibrils with TDP-43 was placed on grids for 5 min, then washed, blocked, and probed following previous protocol²² but with primary antibody using anti-TDP-43 N-term rabbit antibody (10782, 1:1000, Proteintech). In the end, the grids were incubated with an 18 nm gold-conjugated secondary anti-rabbit IgG antibody (1:40, Jackson ImmunoResearch) at RT for 1 h. The unbound antibody was heavily washed by high-salt Tween and PBS. The grids were then fixed by 1% glutaraldehyde in PBS at RT for 10 min and washed six times by double-distilled H₂O. Finally, the grids were negatively stained using 2% uranyl acetate.