Negative Staining of Purified Exosomes: A Procedure to Visualize Exosome Morphology Using Transmission Electron Microscopy
Published: April 30, 2023
Abstract
Source: Jung, M. K. et al. Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy. J. Vis. Exp. (2018)
This video describes a procedure for negative staining of purified exosomes. The negatively stained exosomes are imaged using transmission electron microscopy. This technique helps visualize the morphology of exosomes.
Protocol
1. Negative Staining
- Glow-discharge the thin formvar/carbon film-coated 200 mesh copper EM grids for 1 min by glow discharger.
- 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. - 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.
- Immediately stain with the ~20 drops of filtered 1% uranyl acetate (UA) solution on the surface of the EM grid by syringe.
- Remove the excess UA solution on the grid by contacting the grid edge with filter paper.
- Quickly rinse the grid with a drop of water. This step will remove the excess staining solution.
- 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.
- Store the grid in an EM grid box for future observation by a TEM at 80 kV.
開示
The authors have nothing to disclose.
Materials
| Uranyl acetate | EMS | 22400 | Hazardous chemical |
| Transmission Electron Microscopy | JEOL | JEM2200FS | |
| Glow discharger | JEOL | JFC1100E | |
| Paraformaldehyde | EMS | 19210 | |
| Formvar carbon coated Copper Grid (200 mesh) |
EMS | FCF200-CU | |
| Formvar carbon coated Nickel Grid (200 mesh) |
EMS | FCF200-NI |
