Formvar carbon coated 300 mesh copper grids

A 2:1 mass concentration ratio reaction mix of WT or C291R tau-K18 to heparin was prepared using 0.8 mg/ml of tau-K18 and 0.4 mg/ml of heparin in a total volume of 20 μl. The mixture was incubated at 37°C without shaking for 48 h to form fibrils. Five microliters of each sample were spotted on Formvar carbon-coated 300-mesh copper grids (#AGS162-6, Agar Scientific, Stansted, UK) and allowed 2 min to bind. The unbound sample was removed by blotting with filter paper and 5 μl of uranyl acetate added for 2 min. A JEOL JEM-2010 transmission electron microscope (TEM) was used to image the samples.

Formvar/carbon-coated 300-mesh copper grids (#S162, Agar scientific, UK) were glow-discharged using the ELMO system from Cordouan Technologies. Five microliters of labeled or unlabeled K18 preparations were pipetted onto the grid and allowed to bind for 1 min. Excess samples were removed with a strip of filter paper, and 5 μl of 2% uranyl acetate added for 1 min. After removing the excess stain with a strip of filter paper, the grids were imaged using a JEOL-2100F transmission electron microscope.

Pristine MWNTs, used for preparation of w-MWNTs (diameter 20–30 nm; length 0.5–2.0 μm, batch #1237YJS) were purchased from Nanostructured and Amorphous Materials Inc., USA. Pristine MWNTs used for the preparation of t-MWNTs (diameter 9.5 nm; length 1.5 μm; batch #NC3100) were purchased from Nanocyl s.a., Belgium. Reagents for chemical functionalisation of f-MWNT, PBEC and astrocyte isolation and culturing are listed in SI. Costar Transwell™ permeable 3.0 μm pore polycarbonate supports were purchased from Corning Inc., USA. Chemicals for the Angiopep-2 synthesis, fluoropore PTFE hydrophobic 0.22 μm membrane and glutaraldehyde 25% aqueous solution were purchased from Merck Millipore, UK. The radioactive tracer [¹¹¹In]Cl₃ was obtained from Mallinckrodt Pharmaceuticals (Netherlands) as an aqueous solution in 0.5 M HCl and used without further purification. Carbon/formvar coated 300-mesh copper grids were obtained from Agar Scientific, UK.

The visualization of purified PVY CP-derived VLPs was done with uranyl acetate negative staining. First, 5 µL of the sample (1 mg/mL) was absorbed on carbon formvar-coated 300 Mesh Copper grids (Agar Scientific, Stansted, UK; 2 grids per sample were prepared) and incubated for 3 min. The grids were then washed with 1 mM ethylenediaminetetraacetic acid (EDTA) and negatively stained with 0.5% uranyl acetate aqueous solution. The grids were analyzed with a JEM-1230 electron microscope (JEOL, Tokyo, Japan) at an accelerating voltage of 100 kV and a minimum of five electrograph pictures were made per sample.