Tecnai t12 biotwin

RKO cells were seeded on Nunc Thermanox coverslips (150067; Thermo Fisher Scientific) on a 24-well plate. Furthermore, 24 h after seeding, cells were transfected with 30 nM siRNAs. Then 72 h after transfection, cells were fixed in 2.5% glutaraldehyde for 2 h, then washed three times with PBS, postfixed with 1% osmium tetroxide for 1 h, washed with water, and incubated in 1% aqueous uranyl acetate overnight at 4°C. Then the cells were dehydrated with increasing dilutions of ethanol, infiltrated with epoxy resin (45-359-1EA-F; Sigma-Aldrich), embedded using BEEM capsules (Hanson et al, 2010 (link)), and incubated at 60°C for 72 h. Then polymerized blocks were trimmed and cut with a Leica ultramicrotome (EM UC7) for ultrathin sections (65 nm thick) and collected on copper grids, mesh 300 (AGG2300C; Agar Scientific). Specimen grids were examined with a transmission electron microscope Tecnai T12 BioTwin (FEI) equipped with a 16-megapixel TemCam-F416 (R) camera (TVIPS GmbH).

Cryo-EM images were recorded on an FEI Tecnai T12 Biotwin with LaB₆ source, operating at 120 keV on an FEI Eagle 4k × 4k CCD camera, using a Gatan 626 side entry cryo holder. For correlation with the latest recorded LM image before vitrification, low magnification (<×200) images were recorded by hand, covering the whole grid. Composite image overviews of the whole grid and overlays with the LM images were made in Adobe Photoshop.
Cryo-ET was performed on a Titan Krios (Thermo Fisher Scientific) operated at 300 kV, equipped with a Gatan K3 BioQuantum direct electron detector. Tilt series were recorded using TOMO 4 software (Thermo Fisher Scientific) between −56° to +56° with a tilt step of 2°, starting at 0°, with a total dose of 100 e/Ų and 16 frames per image at a nominal magnification of ×19.500, corresponding to a calibrated pixel size of 4.4 Å/pixel with a defocus of −7 µm. Cryo-electron tomography tilt series were reconstructed using IMOD software^{52 (link),53 (link)}. Surface renderings were performed by hand using Amira software (Thermo Fisher Scientific).

Samples of amplified tau aggregates and amorphous MAP2 were diluted 1:1 with assembly buffer to a final concentration of 2.5 μM. Formvar/carbon-coated 200 mesh copper grids (Electron Microscopy Sciences) were placed for 1.5 min onto 10-μl droplets of these samples. Excess liquid was blotted on Whatman filter paper and the grids were placed for 1.5 min onto 10 μl-droplets of 2% uranyl acetate (Electron Microscopy Sciences, 0.2 μm syringe filtered). Grids were again blotted with filter paper and air-dried for another 5 min. Images were recorded with an FEI Tecnai T12 Biotwin transmission electron microscope at 100 KV using a Gatan CCD camera.

To select cells in metaphase, resin-embedded samples were pre-inspected using an Axiolab RE upright brightfield microscope (Zeiss, Germany) with a 5 x and a 40 x objective lens (Zeiss, Germany). Selected cells in metaphase were sectioned using an EM UC6 ultramicrotome (Leica Microsystems, Austria). Ribbons of semi-thick (~300 nm) serial sections were collected on Formvar-coated copper slot grids, post-stained with 2% (w/v) uranyl acetate in 70% (v/v) methanol, followed by 0.4% (w/v) lead citrate (Science Services, USA) in double-distilled water. In addition, 20 nm-colloidal gold (British Biocell International, UK) was attached to the serial sections, serving as fiducial markers for subsequent electron tomography. The selected cells were then pre-inspected at low magnification (~2900 x) using either an EM906 (Zeiss, Germany) or a TECNAI T12 Biotwin (Thermo Fisher Scientific, USA) transmission electron microscope operated at either 80 or 120 kV, respectively.

The serial sections were imaged using a TECNAI T12 Biotwin transmission electron microscope (Thermo Fisher Scientific, Waltham, MA) operated at 120 kV and equipped with an F214 charge-coupled device (CCD) camera (TVIPS GmbH, Germany). Images of whole cells in metaphase were acquired at 1200× magnification using EMMenu Software (TVIPS GmbH, Germany). To choose a cell for electron tomography, the metaphase plate had to be correctly formed when looking at the chromosome area in 3D and for each chosen cell the half-spindle and the MT angle were measured in the 3D volumes as described (see the Light microscopy section). For this, the EM stacks were projected in 3D. The chromosome and microtubule area were estimated by manually labeling the chromosome and MT area. Both the half-spindle angle and MT angle were finally calculated using the ZIB Amira software (Zuse Institute Berlin, Germany).