Negative Staining of Purified Exosomes: A Procedure to Visualize Exosome Morphology Using Transmission Electron Microscopy

1. Negative Staining

1. Glow-discharge the thin formvar/carbon film-coated 200 mesh copper EM grids for 1 min by glow discharger.
2. Fix purified exosomes with 1 mL of 2% paraformaldehyde (PFA) for 5 min.
   NOTE: Paraformaldehyde fumes are toxic. All work should be done in a ventilated fume hood.
3. Load 5-7 µL exosome suspension solution on the grid and incubate for 1 min. If the concentration of exosome is too high, dilute the concentration to one-half to one-fifth.
4. Immediately stain with the ~20 drops of filtered 1% uranyl acetate (UA) solution on the surface of the EM grid by syringe.
5. Remove the excess UA solution on the grid by contacting the grid edge with filter paper.
6. Quickly rinse the grid with a drop of water. This step will remove the excess staining solution.
7. Place the grid on the table by holding it with tweezers, and cover the grid partially with a culture dish to dry for 10 min under room temperature.
8. Store the grid in an EM grid box for future observation by a TEM at 80 kV.