Tecnai 12 transmission electron microscope

Highly concentrated solutions of ICs (2 mg/ml drug +3 mg/ml ADA) were prepared in PBS. The IC species were separated via SEC with PBS as running buffer. Collected fractions (peak collection, without BSA as additive) with specific IC species (mainly dimers and tetramers) were stored at −20°C. Freshly thawed samples/fractions were diluted in PBS and 4 μl were adsorbed for 60 s to glow-discharged parlodion carbon-coated copper grids. The grids were then blotted, washed with three drops of double-distilled water, incubated with 2 μl of Tobacco Mosaic Virus solution (TMV; kindly supplied by Ruben Diaz-Avalos, New York Structural Biology Center, USA), further washed with two drops of water and finally negatively stained with two drops of 2% uranyl acetate (pH 4.3) solution. Samples were imaged at a nominal magnification of 52,000x using a Tecnai12 transmission electron microscope (FEI, Eindhoven, Netherlands) operating at 120 kV. Electron micrographs were recorded on a 4000 by 4000 pixel charge-coupled device camera (F416, Tietz Video and Image Processing System, Gauting, Germany) yielding a final pixel size of 0.25 nm on the specimen level. Reference-free alignment was performed on manually selected fibril segments from recorded images using the EMAN2 image processing package (15 (link)).

A laboratory-built humidity-controlled vitrification system was used to prepare the samples for Cryo-TEM. Humidity was kept close to 80% for all experiments and ambient temperature was ∼22°C. A 4 μl aliquot of the sample was pipetted onto a 300 mesh copper grid coated with lacy formvar-carbon film (Pro-SciTech, Thuringowa, Queensland). After 30 s adsorption time, the grid was blotted manually using Whatman 541 filter paper, for between 2 and 10 s. The grid was then plunged into liquid ethane cooled by liquid nitrogen. Frozen grids were stored in liquid nitrogen until examined. The samples were examined using a Gatan 626 cryoholder (Gatan, Pleasanton, CA, USA) and a Tecnai 12 Transmission Electron Microscope (FEI, Eindhoven, The Netherlands) at an operating voltage of 120 kV, equipped with a FEI Eagle 4k × 4k CCD (FEI, Eindhoven, the Netherlands). Samples were viewed at 100 000–150 000 times magnification.

Formvar/Carbon Coated - Copper 300 mesh grids (Electron Microscopy Sciences, Hatfield, PA) were glow discharged for 10 s. An aliquot of exosomes (5 µl) isolated from the 10/40 % interface of the sucrose flotation gradient was spread onto a freshly glow- discharged grid. The grid was quickly washed on three droplets of water to dilute the sucrose and then stained with 1 % uranyl acetate for 2 min. Excess staining solution was removed with Whatman Grade one filter paper. Post drying, grids were imaged at 120 kV using a Tecnai-12 Transmission Electron Microscope (FEI, Hillsboro, OR) in the Electron Microscopy Laboratory at UC Berkeley.