Carbon coated 300 meshcopper grids

Carbon-coated 300-mesh
copper grids (Electron Microscopy Sciences, Hatfield) were plasma
cleaned for 5 s using an oxygen plasma cleaner (Zepto RIE, Dienner),
before pipetting 5 μL of Syn oligomers sample (∼100 μg/mL
concentration) in PBS buffer, pH 7.4, on top of the grids followed
by 2 min of incubation. The grids were washed with a water droplet.
Uranyl acetate (2% w/v) was added (3 μL), and the mixture was
incubated for 2 min. Excess stain was blotted off with filter paper
and dried. TEM images were recorded with an FEI Tecnai Spirit operating
at 120 kV. We used ImageJ for particle size analysis of Syn oligomers.^{81 (link)}

Transmission electron microscopy
images were recorded with a FEI Tecnai Spirit operating at 120 kV.
Carbon-coated 300-mesh copper grids (Electron Microscopy Sciences,
Hatfield) were plasma-cleaned for 5 s using an oxygen plasma cleaner
(Zepto RIE, Dienner), and 5 μL of Tau protein (2 μM monomer
concentration) sample in aggregation buffer consisting of 25 mM sodium
phosphate, 25 mM NaCl, 2.5 mM EDTA, and 0.33 mM DTT, pH 6.8, was pipetted
on top of the grids after 100 times dilution in water and incubated
for 2 min. The grids were then washed with a water droplet. Uranyl
acetate (2% w/v) was added (3 μL) and incubated for 2 min. Excess
stain was blotted off with a filter paper and dried. We used ImageJ
to estimate the width of Tau protein fibrils.^{51 (link)}

Full methods used have been published previously^{9 (link)}. Briefly samples were loaded onto carbon-coated 300 mesh copper grids (Electron microscopy Sciences) that were glow discharged for 40 seconds using an PELCO easiGLOW™ glow discharge unit (Ted Pella Inc, USA). Samples were left to bind for 2 minutes, blotted, washed briefly in one water drop, blotted, and then stained with either NanoW stain (Nanoprobes) 2 × 1 min or 2% (w/v) uranyl acetate in water for 45 sec, then blotted and air-dried. Grids were inserted into the microscope using a dedicated sample holder for mouse prions with strict adherence to risk assessment and microbiological containment level 2 safe working practice. The sample holder was decontaminated directly after use by exposing to plasma using a Fischione 1020 plasma cleaner. Images were acquired on an FEI Tecnai T10 electron microscope (FEI, Eindhoven, NL) operating at 100 kV and recorded on a 1 k × 1 k charged couple device (CCD) camera (Gatan) at a nominal magnification of 44,000 with a pixel size of 3.96 Å. Fibril dimensions were measured in ImageJ^{41 (link)} and IMOD^{42 (link)}. Because of their helical twist, prion rods and recombinant PrP fibrils viewed on a surface alternate between wider, face-on and narrower, edge-on views of the structure. The dimensions reported here were measured on the widest parts of the fibrils.