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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 (from Thermo Fisher Scientific)

  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掳C in 2 to 3 mL of ice-cold 0.1 渭m 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 碌L of suspension for each TEM grid. The concentration should be around 1 to 5 渭g/渭L.
  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.

Optimized Protocol for Negative Staining of Extracellular Vesicles and Particles (EVPs)

1. Grid Preparation

  • 鲍蝉别听carbon-coated 200-mesh Cu TEM grids, carbon side up.
  • Place grids in the听Pelco easiGlow听with the metal grid holder.
  • Glow discharge听蹿辞谤听30 seconds at 20 碌A听to render the surface hydrophilic.
  • Use grids听within 20 minutes听of glow discharge.

2. Sample Preparation

  • 笔谤别辫补谤别听at least two grids per specimen听when possible.
  • If the sample is too concentrated,听dilute with PBS or dH鈧侽听to a final concentration of听0.1 渭g/渭L.

    鈿狅笍听Note: Avoid phosphate buffers as they cause uranyl acetate precipitation.

3. Sample Application

Place a clean sheet of听parafilm听on the glass plate at the听negative staining station (Room SADB B/6).

Choose one of the following methods:

Method 1: Direct Application

  • Using self-locking tweezers, pipette听5鈥�10 碌L听of EVP solution onto the听carbon side听of the grid.
  • Incubate for听5 minutes.

Method 2: Drop Incubation

  • Pipette a听20 碌L听drop of EVP solution onto parafilm.
  • Place the grid听carbon side down听on the drop.
  • Incubate for听5鈥�10 minutes, covering with a lid to prevent disturbance.
  • To increase EVP concentration:
    • Incubate for 10 minutes.
    • Remove the grid briefly.
    • Reapply to the same drop for additional accumulation.

4. Washing and Staining

Glycine Wash

  • Transfer the grid (sample side down) onto听three 50 碌L drops of 0.2 M glycine,听2 minutes each.

Water Wash

  • Transfer to听five 100 碌L drops of ddH鈧侽,听1 minute each.

Negative Staining

  • Place the grid (sample side down) on a听20 碌L drop of filtered 2% uranyl acetate听蹿辞谤听1 minute.

    鈿狅笍听Note: Uranyl acetate is acidic; avoid for acid-sensitive particles.

  • 骋别苍迟濒测听wick off excess stain听using the edge of filter paper.

5. Drying and Storage

  • Allow the grid to听air dry for 60 minutes at room temperature, or use a听heat lamp.
  • Proceed to听TEM imaging听or store in a听grid box听for up to听1鈥�2 weeks.

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Common mistakes or pitfalls听that can occur when following the negative staining protocol for extracellular vesicles and particles (EVPs), along with tips to avoid them:

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馃敩听1. Improper Glow Discharge

  • Mistake:听Not using the carbon side up or exceeding the 20-minute window post-discharge.
  • Impact:听Leads to poor sample adherence and uneven distribution.
  • Tip:听Always confirm the carbon side is facing up and time your sample application promptly.

馃挧听2. Sample Overload or Underload

  • Mistake:听Applying too concentrated or too dilute a sample.
  • Impact:听Overload causes clumping; underload results in too few particles for imaging.
  • Tip:听Adjust concentration to ~0.1 渭g/渭L and visually inspect density before staining.

馃И听3. Using Phosphate Buffers

  • Mistake:听Using PBS or other phosphate-containing buffers with uranyl acetate.
  • Impact:听Causes precipitation and artifacts on the grid.
  • Tip:听Use dH鈧侽 for dilution and rinsing before staining.

馃晵听4. Inconsistent Incubation Times

  • Mistake:听Varying incubation times between grids or skipping the reapplication step in Method 2.
  • Impact:听Leads to inconsistent EVP density across grids.
  • Tip:听Use a timer and standardize incubation steps across all samples.

馃Щ听5. Improper Wicking Technique

  • Mistake:听Touching the sample area directly with filter paper.
  • Impact:听Can remove or damage the sample.
  • Tip:听Wick from the听edge听of the grid only.

馃Т听6. Inadequate Washing

  • Mistake:听Skipping or shortening glycine and water washes.
  • Impact:听Residual fixatives or salts can interfere with staining and imaging.
  • Tip:听Follow the full wash sequence to ensure clean background and contrast.

鈽笍听7. Inappropriate Use of Uranyl Acetate

  • Mistake:听Using uranyl acetate on acid-sensitive particles.
  • Impact:听Particle degradation or morphological changes.
  • Tip:听Consider alternative stains (e.g., ammonium molybdate) for acid-sensitive samples.

馃尅锔徧�8. Incomplete Drying

  • Mistake:听Imaging before the grid is fully dry.
  • Impact:听Can cause beam damage or poor image quality.
  • Tip:听Ensure at least 60 minutes of drying or use a heat lamp if needed.

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 碌A.
  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 碌L 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 碌L of a 0.2 M glycine solution, each for 3 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 碌L of primary antibody (e.g., anti-PD-L1) diluted in PBS with 0.1% 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鈥檚 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 碌L of secondary antibody (dilution 1:20 with 0.1% BSA) 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. Transfer the grid sample side down onto a drop of 1% glutaraldehyde in 0.1M sodium cacodylate buffer for 2 minutes.
  15. Wash the grid sample side down with five separate drops of ddH2O for 1 min each.
  16. 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 occurs during drying and under vacuum.

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