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Negative Staining

Adapted and Prepared by Jeannie Mui

Negative Staining and Immunogold Labelling 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

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Negative Staining and Immunogold Labelling of EVPs

  1. Fix the purified EVPs or exosomes following isolation with 4% paraformaldehyde in 0.1 M phosphate buffer solution at a 1:1 ratio. Gently mix and resuspend the exosome solution and bring it to the negative staining station at the FEMR (Room B/5) within one to two hours.
  2. Glow discharge 200-mesh Cu TEM grids with carbon film side up for 30 seconds at 20 µA.
  3. Place a large piece of Parafilm on the glass plate at the negative staining station in room B/5.
  4. Load five to seven µL of the EVP solution onto the carbon side of the TEM grid and incubate for 15 minutes.
  5. Float the grid sample side down onto three separate drops of 100 uL of PBS, each for 5 minutes.
  6. Float the grid sample side down onto three separate drops of 50 µL of a 0.2 M glycine solution, each for 5 minutes to quench the free aldehyde groups.
  7. Transfer the grids sample side down onto a drop of PBS containing 1% BSA and block for 5 minutes.
  8. Incubate the grids with 20 µL of primary antibody (e.g., anti-PD-L1) diluted in PBS with 1% BSA for one hour at room temperature or overnight at 4oC inside a humid chamber.
  9. Wash the grid sample side down with five separate drops of Dulbecco’s PBS (DPBS) for 5 minutes each.
  10. Transfer the grid sample side down onto a drop of PBS containing 1% BSA and block for 5 minutes.
  11. Incubate the grids with 20 µL of secondary antibody (dilution 1:20) for one hour.
  12. Wash the grid sample side down with five separate drops of DPBS for 5 minutes each.
  13. Wash the grid sample side down with five separate drops of ddH2O for 2 min each.
  14. Perform negative staining with a 2% uranyl acetate solution (as described in the steps above), and image the samples by TEM, or store them in a TEM grid box for future observation. The exosome morphology observed is the product of negative staining with nanogold particles. Exosomes typically show a cup-shaped morphology, an artifact that can occur during drying or under vacuum.

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