300 mesh copper grid

The drop-on-the-grid method (DEG) was used as follows. A drop (10 µL) of sample suspension containing 10⁵ Vaccinia viruses was fixated by incubation with 2.5% paraformaldehyde (Electron Microscopy Sciences, PA, USA) for 30 min at rt followed by another 30 min of incubation at 37 °C. Then, the sample was placed directly onto a glow-discharged EM sample support, 300 MESH copper grid, covered with carbon film (Electron Microscopy Sciences, PA, USA). After adsorption for 10 min at room temperature, the grid was washed three times in double-distilled water and negatively stained with 1% phosphotungstic acid, pH 4.5. The grids were examined using a TECNAI T12 FEI (ThermoFisher, OR, USA) transmission electron microscope operated at 200 kV. Micrographs were recorded using an Erlangsheng 782 ES 500W camera (Gatan, CA, USA) at a resolution of 2048 × 2048 pixels.

Ten microliters of exosome encapsulation product was covered by a support film of a 300-mesh copper grid (Electron Microscopy Sciences, USA) for 20 min of absorption. After 3 min of washing with water three times, uranyl–acetate solution was used to incubate the grids for 10 min. The grids were then washed with water three times to complete negative staining. Finally, an electron microscope (FEI Company, USA) was used to image the grids.

For the analysis of structure and morphology of samples at different Aβ42 aggregation time-points, 5 μL aliquots directly removed from the aggregation plate were adsorbed into carbon-coated collodion film supported on 300-mesh copper grids (Electron Microscopy Sciences, PA, USA) and negatively stained twice with 1% (m/v) uranyl acetate (Electron Microscopy Sciences, PA, USA). Grids were visualized with a JEOL (Tokyo, Japan) JEM-1400 transmission electron microscope equipped with an Orious (CA, USA) Sc1000 digital camera, and exhaustively observed.

Transmission electron microscopy (TEM) was performed by using a transmission electron microscope (JEM-1400 Plus, JEOL, Tokyo, Japan), working at 120 kV. Images of NPs were obtained by placing a drop of the corresponding suspension on 300-mesh copper grids coated with carbon from Electron Microscopy Science (Hatfield, PA, USA). These samples were left to dry before being placed under the microscope.

EVs (50 μg/mL of total protein) were placed on 300-mesh copper grids (Electron Microscopy Sciences, Hatfield, USA) and stained with 2% uranyl acetate for 12 h. Images were obtained by using a JEM1011 microscope (JEOL, Akishima, Japan) at an accelerating voltage of 100 kV.

Transmission electron microscopy (MET, JOEL model JEM 10–10) was used to morphometrically visualize cell-wall thickening in the VT-MRSA isolates as an effect of antibiotic concentration. Vancomycin-treated bacteria were grown to the exponential phase in the presence of gradual increments of 1 mg/mL antibiotics to a final concentration of 20 μg/mL. As a control, untreated bacteria were also grown to the exponential phase. The bacteria were harvested, washed, and fixed with a glutaraldehyde/formalin (2.5%/10%) solution in 0.1 M phosphate-buffered saline (PBS), pH 6.0. Subsequently, the bacteria were post-fixed with osmium tetroxide, contrasted with uranyl acetate, and dehydrated in graded concentrations of ethyl alcohol (20, 30, 40, 50, 60, 70, 80, 90, and 100%). Then, transverse thin sections from samples embedded in resin were mounted on grids (300-mesh copper grids; Electron Microscopy Sciences, Hatfield Pennsylvania), followed by treatment with lead citrate. The samples were stained with 1% phosphotungstic acid at pH 7.2 and visualized by TEM. The images were processed at 100,000x and analyzed to determine the cell-wall thickness from an average of 10 cells per bacterial strain. The S. aureus strain ATCC 25923 and the VISA strain Mu50 ATCC 700699 were used as references.