Tecnai spirit em

Samples were analyzed by negative-stain electron microscopy (EM). A 3-μl aliquot containing ∼0.05 mg/ml of the trimer was applied for 25 s onto a carbon-coated 400-mesh Cu grid that had been glow discharged at 20 mA for 30 s. The grid was then negatively stained with 2% uranyl formate for 30 s. Data were collected using a FEI Tecnai Spirit EM operating at 120 kV, with an electron dose of ∼29 e⁻/Ų and a magnification of ×52,000 that resulted in a pixel size of 2.05 Å at the specimen plane. Images were acquired with a Tietz 4k × 4k TemCam-F416 CMOS camera using a nominal defocus of 1,000 nm and the Leginon package (51 (link)).

Cells were grown on MatTek glass-bottomed Petri dishes and fixed in 2.5% glutaraldehyde 2% paraformaldehyde in 0.1 M cacodylate buffer pH7.4 O/N at 4°C. Washed in buffer and postfixed in 1% osmium tetroxide for 1 h at 4°C. Dehydrated in an ascending ethanol series and embedded in CY212 resin. Ultrathin sections were stained with saturated aqueous uranyl acetate and Reynolds lead citrate and examined using a Tecnai Spirit EM (FEI) operated at 80 KV.

5 μl of samples were applied on glow discharged, carbon-coated copper EM grids for 1min. The excess sample was then washed off by dipping the grid in milli-Q water. The sample on the grid was then fixed/negatively stained with 2% Uranyl acetate for 30 sec and then the excess fluid was removed by using filter paper. The samples were imaged at a nominal magnification of 63,000x or 87,000x on a FEI Tecnai Spirit EM operated at 80eV and fitted with a digital camera. MT length and number of MT bundles were quantified from images obtained from three independent experiments.