Transmission Electron Microscopy of PrP Fibrils

The formation of fibrils by PrPs was confirmed by electron microscopy of negatively stained samples. The preparation for negatively stained samples was described in detail previously [28] (link), [33] (link)–[35] (link). Briefly, the incubation time was chosen within a time range of the plateau of each kinetic curve of ThT fluorescence shown in Fig. 1. Sample aliquots of 10 µl were placed on copper grids and left at room temperature for 1–2 min, rinsed twice with H₂O, and then stained with 2% (w/v) uranyl acetate for another 1–2 min. The stained samples were examined using an FEI Tecnai G2 20 transmission electron microscope (Hillsboro, OR) operating at 200 kV or an H-8100 transmission electron microscope (Hitachi, Tokyo, Japan) operating at 100 kV.