Talos l120c g2 transmission electron microscope

For the TEM analyses, the ET samples were negatively stained by depositing a sample drop on a TEM grid covered with a formvar film. The excess drop was removed after 1 min from the grid with filter paper to keep a light veil of the sample on the supporting substrate. A drop of 2% phosphotungstic acid was placed on the grid for 1 min and then removed with filter paper to surround the nanosystems deposited on the grid and adhere to their surface. Then, the grid was observed with a TALOS L120C G2 Transmission Electron Microscope (Thermo Fisher Scientific, Eindhoven, The Nederlands).

Tau samples were prepared as follows, 20 μM tau and 10 μM 3CL^pro were incubated for 72 h at 37°C. To prepare specimens for negative staining TEM, a total volume of 3 μL from the corresponding sample was pipetted onto carbon coated 300 Mesh copper EM grids, which were operated by anti-capillary tweezers and glow discharged in advance for 90 seconds using a PELCO easiGlow^™ (Ted Pella, Inc., Redding, California, USA) at 0.39 mbar and 15 mA. After an incubation time of 1 min excess liquid was blotted away by touching the edge of the grid with filter paper. Subsequently the grids were stained with two 4 μL droplets of a 2% Uranyl acetate solution. While the first droplet’s excess liquid was immediately blotted after application, the second droplet stayed on the grid for an incubation time of 1 min before blotting. Finally, the grids were left to dry at room temperature for approximately 1 min. These negatively stained samples were examined on a Talos L120C G2 transmission electron microscope (Thermo Fisher Scientific, Waltham, Massachusetts, USA) which was operated at 120 kV (LaB6 (Lanthanum hexaboride)/Denka). Micrographs were collected in medium magnification (6700x) and high magnification (73kx) on a 4k x 4k Ceta 16M CEMOS camera using the Velox^™ software (Thermo Fisher Scientific, Waltham, Massachusetts, USA).

For ultrastructural analysis, purified synaptosomes were fixed in 2.5% glutaraldehyde in 0.1M cacodylate buffer, pH 7.2, post-fixed in 1% osmium tetroxide in cacodylate buffer 0.1 M, pH 7.2, en bloc stained with a 1% aqueous solution of uranyl acetate. The dehydration was performed through a graded ethanol series. Samples were then embedded in LX112 (Polysciences Inc., Warrington, PA, USA), polymerized for 12 h at 42 °C, followed by 48 h at 60 °C. A Leica Ultracut E microtome was used to prepare grey-silver ultrathin sections that were then stained with uranyl acetate and lead citrate. All images were acquired using a FEI Talos L120C G2 Transmission Electron Microscope (Thermo Scientific™, Monza, Italy).