Jem 2200fs cr

Fecal, spleen and the infected cell culture samples were processed for EM observation as previously described[7 (link)]. Briefly, fecal and spleen samples were processed in 1:10 (w/v) with 10 mM, pH7.2 PBS, and homogenized before centrifuging at 8000 × g for 20 min at 4°C. The infected cells were frozen and thawed for 3 times before centrifugation as described above. The supernatants were then incubated with 1% phosphotungstic acid, and the viruses were examined using electron microscope (JEM-2200FS/CR)(JEOL, Tokyo, Japan).

For cryo-electron microscopy, uEV preparations were directly adsorbed onto glow-discharged holey carbon grids (100 Holey carbon film of Cu with mess 200; Quantifoil^®, Germany). Grids were blotted at 95% humidity and rapidly plunged into liquid ethane with the aid of VITROBOT (Maastricht Instruments BV, The Netherlands). Vitrified samples were imaged at liquid nitrogen temperature using a JEM-2200FS/CR transmission cryo-electron microscope (JEOL, Japan) equipped with a field emission gun and operated at an acceleration voltage of 200 kV.

For sample preparation, a freshly glow-discharged 300-mesh only–carbon coated grid (carbon film on copper 300 mesh; C300Cu100; EM Resolutions) is placed inside the chamber of a Vitrobot Mark II (FEI Company) at 8 °C for 30 s followed by fast plunging into liquid ethane. cryo-EM was performed on a JEM-2200FS/CR (JEOL Europe, Croissy-sur-Seine, France) transmission electron microscope equipped with a field emission gun operated at 200 kV and an in-column Ω energy filter. Digital images were recorded on a 4,000 × 4,000 (15 μm pixels) Ultrascan4000^TM charge-coupled device camera (Gatan Inc.) using DigitalMicrograph^TM (Gatan Inc.) software, at a nominal magnification of 60,000×, resulting in a final sampling of 1.7 Å/pixel.

The cryo-electron microscopy (EM) was performed following the protocol used by Perez and colleagues (29 (link)). Briefly, 10 μl of EV preparations were directly adsorbed onto glow-discharged holey carbon grids (QUANTIFOIL Micro Tools GmbH). Grids were blotted at 95% of humidity and rapidly plunged into liquid ethane with the aid of VITROBOT (Maastricht Instruments B). Vitrified samples were imaged at liquid nitrogen temperature using a JEM-2200FS/CR transmission cryo-electron microscope (JEOL) equipped with a field emission gun and operated at an acceleration voltage of 200 kV.

All samples were prepared in 10 mM buffer phosphate (pH 7.4) with 16% of DMSO. Cryo-TEM data were collected on a JEM-2200FS/CR transmission electron microscope (JEOL, Japan), equipped with an UltraScan 4000 SP (4008 × 4008 pixels) cooled slow-scan CCD camera (GATAN, UK). Three microliters of the compound were vitrified on Quantifoil 2/2 grids, using Vitrobot (FEI) and were analyzed at nitrogen liquid temperature with a TEM operated at 200 kV in low dose conditions.

For negative staining, vesicles were adsorbed onto glow-discharged Formvar-Carbon Niquel grids, washed with distilled water and stained with freshly prepared 2% uranyl acetate in aqueous suspension. Negative stained samples were imaged at room temperature using a JEM-1230 transmission electron microscope (JEOL) equipped with a thermionic tungsten filament and operated at an acceleration voltage of 120 kV. Images were taken using the ORIUS SC1000 (4008 × 2672 pixels) cooled slow-scan CCD camera (GATAN). For cryo-electron microscopy, EV preparations were directly adsorbed onto glow-discharged holey carbon grids (QUANTIFOIL, Germany). Grids were blotted at 95% humidity and rapidly plunged into liquid ethane with the aid of a VITROBOT (Maastricht Instruments BV, The Netherlands). Vitrified samples were imaged at liquid nitrogen temperature using a JEM-2200FS/CR transmission cryo-electron microscope (JEOL, Japan) equipped with a field emission gun and operated at an acceleration voltage of 200 kV.

For
2D cryo-imaging, 4 μL of sample (0.5 mg/mL) was applied directly
onto a glow-discharged 200-mesh Quantifoil R 2/2 holey-carbon grid
and rapidly plunged into liquid ethane with the help of a Vitrobot
Mark III (FEI Inc., Eindhoven, The Netherlands). Sample analysis at
liquid nitrogen temperature was carried out with a JEM-2200 FS/CR
(JEOL Ltd.) transmission electron microscope, using an acceleration
voltage of 200 kV and defocus ranging from −1.5 to −5.0
μm. Images were taken under low-dose conditions on a 4K ×
4K UltraScan 4000 CCD camera (Gatan Inc., Pleasanton, CA, USA). An
in-column Omega energy filter was used in the microscope with the
energy slit width set at 30 eV, to improve the signal-to-noise ratio
of the images. Average sizes and dimensions were determined with ImageJ
software (version 1.51j8) by measuring the line intensity profile
of a representative number of assemblies.