H 8100 transmission electron microscope

The macaque SEVI was generated through overnight agitation of macaque PAP248-286 at 37°C and 1400 rpm by a thermomixer (Eppendorf, Germany). The macaque SEVI fibrils were characterized by Congo-red staining, ThT fluorescence assay ^{27 (link)} and transmission electron microscopy (TEM) ^{19 (link)}. Briefly, 5 μL of amyloid fibrils from macaque SEVI (1 mg/mL) with or without EGCG treatment were incubated with 100 μL of Congo-red solution (20 μg/mL in PBS) for 10 min at room temperature. The mixture was centrifuged for 5 min at 14,000 rpm and the supernatant was removed. The pellets were dissolved in 100 μL DMSO and the absorbance was read at 490-650 nm in a SpectraMax i3x microplate reader (Molecular Devices, Sunnyvale, CA) ^{6 (link)}. For ThT fluorescence assay, 5 μL of macaque SEVI (1 mg/mL) with or without EGCG treatment were mixed with 95 μL of ThT (50 μM in PBS). The vortexed mixture was analyzed for the fluorescence intensity was measured by excitation at 440 nm and emission at 482 nm ^{27 (link)}. For characterization of the fibrils by TEM, suspension of macaque SEVI-amyloid fibrils (100 μg/mL) in the presence or absence of EGCG (100 μM) were dropped onto the 200-mesh carbon-coated copper grids for 5 min. The grids were then stained with 2% aqueous uranyl acetate for 2 min. Fibrils were imaged by H-8100 transmission electron microscope (Hitachi, Tokyo, Japan) ^{19 (link)}.