T12 electron microscope

Samples were spotted onto non-holey grids and left for 160 to 180 s. Remaining liquid was wicked off and then left to dry before analyzing. Samples for negative-stain TEM were treated with 2% uranyl acetate after sample was wicked off the grid. After 1 min, the uranyl acetate was wicked off. The grids were analyzed using a T12 Electron Microscope (FEI, Hillsboro, OR). Images were collected at 3200x or 24,000x magnification and recorded using a Gatan 2k × 2 k CCD camera.

Images were taken under low dose conditions (20 e⁻/ Ų) with a FEI T12 electron microscope (FEI, Hillsboro, OR, USA) at 120 kV using an ORIUS SC1000 camera (Gatan, Inc., Pleasanton, CA, USA). Samples were prepared according to two protocols: TEM1, the carbon flotation technique [56 (link),57 ] or, TEM2, directly deposited on carbon-coated grid (S160-4 grids, Agar Scientific Ltd, Stansted, UK). For the flotation technique, 4 μL of samples were injected to the clean side of carbon protected by a mica surface; the carbon was separated from mica by floating on a water drop; and a grid was placed on top of the carbon film, which was subsequently air-dried. The substrate carbon-coated mica was produced by evaporation of a carbon in an Emitech K950X carbon coater (QUORUM Technologie Ltd., Asford, UK). For the carbon-coated technique, samples were directly deposited on the carbon-coated grid, the excess of sample was first removed by blotting with a filter paper and then air-dried. Instrument calibration was performed during regular maintenance from the manufacturer. Raw dm3 images (4008 × 2672 px) were analyzed with Gwyddion using cross-section profiles.

The fibril sample (3 μL) was spotted onto a freshly glow-discharged carbon-coated electron microscopy grid. After 1 min, 6 μL uranyl acetate (2% in aqueous solution) was applied to the grid for 2 min. The excessive stain was removed by a filter paper. The samples were imaged using an FEI T12 electron microscope.

Exosome extraction was performed according to the protocol of the Invitrogen Total Exosome Isolation kit (catalog number: 4484453). We used electron microscopy to identify the shape and size of the substances extracted and to confirm isolation of exosomes. The extracted exosomes were resuspended in 1× PBS. Then, aliquots (5 µL) of the exosome samples were placed on carbon-coated grids (previously treated with plasma cleaner; Ted Pella Inc, CA, USA). The samples were blotted with filter paper after 30 s. Then the samples were stained with 2% uranyl acetate for 1 min. The grids were examined under the FEI T12 electron microscope at 120 kV. The micrographs were taken using a Gatanultra scan 4K × 4K.

Copper grids for negative stain were prepared by the carbon flotation technique. Samples were diluted to ~ 0.05 mg/mL in 20 mM Tris (pH 8.0) and small aliquots were adsorbed on carbon-coated mica. The mica was then transferred to a staining solution containing 2% (w/v) sodium silico tungstate, causing detachment of the carbon film. Subsequently, a copper grid was placed on top of the detached carbon which was recovered and dried under air flow. Images were taken under low dose conditions at a nominal magnification of 23,000 × or 30,000 × with a T12 electron microscope (FEI, Hillsboro, OR) at an operating voltage of 120 kV using an ORIUS SC1000 camera (Gatan, Inc., Pleasanton, CA).

Protein samples were pipetted onto the surface of carbon coated copper grids and stained with 1% (w/v) uranyl acetate. Images were taken on an FEI T12 electron microscope.