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 4^oC), 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 4^oC.
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

• Use carbon-coated 200-mesh Cu TEM grids, carbon side up.
• Place grids in the Pelco easiGlow with the metal grid holder.
• Glow discharge for 30 seconds at 20 µA to render the surface hydrophilic.
• Use grids within 20 minutes of glow discharge.

2. Sample Preparation

• Prepare at least two grids per specimen when possible.
• If the sample is too concentrated, dilute with PBS or dH₂O to a final concentration of 0.1 μg/μL.

  ⚠️ Note: Avoid phosphate buffers as they cause uranyl acetate precipitation.

3. Sample Application

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

Choose one of the following methods:

Method 1: Direct Application

• Using self-locking tweezers, pipette 5–10 µL of EVP solution onto the carbon side of the grid.
• Incubate for 5 minutes.

Method 2: Drop Incubation

• Pipette a 20 µL drop of EVP solution onto parafilm.
• Place the grid carbon side down on the drop.
• Incubate for 5–10 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 three 50 µL drops of 0.2 M glycine, 2 minutes each.

Water Wash

• Transfer to five 100 µL drops of ddH₂O, 1 minute each.

Negative Staining

• Place the grid (sample side down) on a 20 µL drop of filtered 2% uranyl acetate for 1 minute.

  ⚠️ Note: Uranyl acetate is acidic; avoid for acid-sensitive particles.

• Gently wick off excess stain using the edge of filter paper.

5. Drying and Storage

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

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

🔬 1. Improper Glow Discharge

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

💧 2. Sample Overload or Underload

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

🧪 3. Using Phosphate Buffers

• Mistake: Using PBS or other phosphate-containing buffers with uranyl acetate.
• Impact: Causes precipitation and artifacts on the grid.
• Tip: Use dH₂O for dilution and rinsing before staining.

🕒 4. Inconsistent Incubation Times

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

🧻 5. Improper Wicking Technique

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

🧴 6. Inadequate Washing

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

☢️ 7. Inappropriate Use of Uranyl Acetate

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

🌡️ 8. Incomplete Drying

• Mistake: Imaging before the grid is fully dry.
• Impact: Can cause beam damage or poor image quality.
• Tip: 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 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 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 µL of primary antibody (e.g., anti-PD-L1) diluted in PBS with 0.1% BSA for one hour at room temperature or overnight at 4^oC 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 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.