McGill.CA / Facility for Electron Microscopy Research / Resources / Protocols

Negative Staining

Adapted and Prepared by Jeannie Mui

Negative Staining of Extracellular Vesicles and Particles (EVPs)

Ultracentrifugation protocol

  1. Collect your EVPs by ultracentrifugation or other means. For ultracentrifugation, spin as needed (e.g., ~120,000 g for 80 to 90 min at 4oC), pour off supernatant and add conditioned media until the entire volume is processed into a pellet.
  2. Wash the pellet for 30 min at 4°C in 2 to 3 mL of ice-cold 0.1 μm sterile-filtered 1X PBS on a shaker, followed by another spin and one final wash/spin to remove secreted proteins and other components.
  3. Resuspend pellet in 2.5% glutaraldehyde fixative solution in 0.1M sodium cacodylate buffer (final pellet resuspension depends on how much is the starting material, the number of TEM grids you want to use, and the amount of EVPs in your sample, 5 to 10 µL of suspension for each TEM grid. The concentration should be around 1 to 5 μg/μL.
  4. Transfer EVPs to a 1.5 mL Eppendorf test tube and store them at 4oC.
  5. Bring to the FEMR for negative staining and TEM imaging within one to two days.


Optimization of negative staining of extracellular vesicles and particles (EVPs)

  1. Using the Pelco easiGlow and the metal grid holder, the glow discharge carbon-coated 200-mesh Cu TEM grids with the carbon film side up for 30 seconds at 20 µA to make the surface more hydrophilic and ensure even distribution of the sample across the grid. Use the grids within 20 minutes.
  2. When possible, prepare at least two grids per specimen. If the material on the TEM grid is too dense, dilute the specimen with PBS or dH2O to a final concentration of 0.1 μg/μL. Note: uranyl acetate precipitates in the presence of phosphate salts.
  3. Place a small sheet of parafilm on the glass plate at the negative staining station in room SADB B/6.
  4. Use either method below.
    1. Method 1: Using self-locking tweezers, pipette a 5 to 10 µL drop of the pre-enriched EVP solution onto the carbon film side of the TEM grid and incubate for 5 minutes.
    2. Method 2: Pipette a 20 mL drop of pre-enriched EVP solution onto the parafilm. Transfer the TEM grid, carbon film side down, onto the EV drop for 5 to 10 minutes. Cover the grid during incubation with a lid to avoid disturbing the grid. The concentration of EVPs on a grid can be increased by incubating the grid on the drop of EVs for 10 minutes, removing the grid for a few minutes, and then putting the grid back on the same drop of EVPs. The EVPs will accumulate at the water-air interface and can be picked up by the grid in several steps.
  1. Transfer the grid, sample-side down, onto separate 50 µL drops of a 0.2 M glycine solution 3 x 2 min.
  2. Transfer the grid with sample-side down onto separate 100 µL drops of dH2O 3 x 1 min.
  3. Transfer the grid with sample-side down onto one 20 uL drop of filtered 2% uranyl acetate solution for 1 minute. Because 2% uranyl acetate has a low pH, it is not recommended for particles that are unstable in acidic conditions.
  4. Wick away the excess uranyl acetate solution from the grid by touching the grid edge with filter paper.
  5. Allow the grid to dry for 15 to 30 minutes at room temperature (or use a heat lamp), and then image by TEM or store it in a grid box for future observation (within one to two weeks).

See also https://www.youtube.com/watch?v=wffPeZMejm0.

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