300 mesh carbon coated copper grid

To further characterize the EV obtained from GBM neurospheres and to confirm their ultrastructural morphology, transmission electron microscopy (TEM) was performed on EV. After collection, EV were resuspended and diluited in PBS and, according to proper dilutions, the samples were adsorbed onto 300-mesh carbon-coated copper grids (Electron Microscopy Sciences) for 5 min in a humidified chamber at room temperature. EV on grids were fixed in 2% glutaraldehyde (Electron Microscopy Sciences) in PBS for 10 min and then briefly rinsed in Milli-Qwater. Grids with adhered EV were examined with a Zeiss Gemini SEM 500 equipped with a STEM detector at 20 kV and at a 3.0 mm working distance, after negative staining with 2% phosphotungstic acid, brought to pH 7.0 with NaOH [62 (link)].

Aliquots (3 μL) of the peak procapsid-containing fractions and phage fractions from the sucrose gradients of the cell lysates (prepared above, Section 2.4) were adsorbed onto 300-mesh carbon-coated copper grids (Electron Microscopy Sciences, Hatfield, PA, USA) for 1 min at room temperature (~22 °C). The grids were washed with distilled water and stained with 1% uranyl acetate for 30 s at room temperature. The grids were blotted to remove excess stain and visualized in a FEI TecnaiG2 Spirit BioTWIN TEM at 68,000× magnification and 80 kV.

Transmission electron microscopy (TEM) was performed on isolated EVs and resuspended in PBS to analyze their ultrastructural morphology. According to proper dilutions, the samples were adsorbed to 300 mesh carbon-coated copper grids (Electron Microscopy Sciences) for 5 min. in a humidified chamber at room temperature. The EVs on grids were then fixed in 2% glutaraldehyde (Electron Microscopy Sciences) in PBS for 10 min. and then briefly rinsed in Milli-Qwater. The grids with adhered EVs were examined with a Zeiss Gemini SEM 500 equipped with a STEM detector at 20 kV and at a 3.0 mm working distance, after negative staining with 2% phosphotungstic acid, brought to pH 7.0 with NaOH [51 (link)].

Negative staining electron microscopy was performed on tau fibrils. 2 μL of tau fibrils (1 mg/mL) were applied to 300 mesh carbon coated copper grids (Electron Microscopy Sciences, Hatfield, PA) and were allowed to settle for 10 min. Grids were washed with filtered PBS. 1% uranyl acetate was filtered through a 2-micron filter and applied to each grid that were then dried. Samples were examined with a FEI Tecnai G2 Spirit Twin TEM (FEI Corp., Hillsboro, OR) operated at 120 kV and digital images were acquired with a Gatan UltraScan 2 k × 2 k camera and Digital Micrograph software (Gatan Inc., Pleasanton, CA).

Electron microscopy analysis was performed as already described [10 (link)]. Briefly, for scanning electron microscopy (SEM) the cells were fixed in PBS (Sigma-Aldrich) 2% glutaraldehyde (Sigma-Aldrich) and dehydrated with ethanol solutions. Next, samples were sputter-coated with an SCD040 Balzer Sputterer (Oerlikon Balzers, Balzers, Liechtenstein). Samples were analyzed with an SEM Philips 505 microscope (Philips, Amsterdam, Netherlands). Transmission electron microscopy was performed on concentrated EV preparations resuspended in PBS (Sigma-Aldrich) to analyze their ultrastructural morphology. According to proper dilutions, the samples were adsorbed to 300 mesh carbon-coated copper grids (Electron Microscopy Sciences) for 5 min in a humidified chamber at room temperature. EVs on grids were then fixed in 2% glutaraldehyde (Sigma-Aldrich) in PBS for 10 min and then briefly rinsed in milli-Q water. Grids with adhered EV were examined with a Philips CM 100 transmission electron microscope TEM at 80 kV, after negative staining with 2% phosphotungstic acid, brought to pH 7.0 with NaOH. Images were captured by a Kodak digital camera.