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Negative Staining

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

Adapted and Prepared by Jeannie Mui
Revised for FEMR Users

Purpose

This protocol describes the preparation of extracellular vesicles and particles (EVPs), including exosomes and microvesicles, for transmission electron microscopy (TEM) using negative staining. The procedure is designed to maximize particle preservation, improve particle adsorption to TEM grids, and minimize staining artifacts.

A. EVP Isolation by Ultracentrifugation

Sample Collection

EVPs may be isolated by ultracentrifugation or other validated purification methods.

  1. Centrifuge conditioned medium as required for your isolation protocol (typically approximately 120,000 × g for 80 to 90 minutes at 4°C).
  2. Carefully remove the supernatant without disturbing the pellet.
  3. Resuspend the pellet in fresh conditioned medium or buffer and repeat centrifugation until the desired sample volume has been processed.
  4. Following the final centrifugation step, remove the supernatant completely.

Pellet Washing

To remove contaminating proteins, soluble macromolecules, and media components:

  1. Gently resuspend the pellet in 2 to 3 mL of ice-cold, sterile-filtered (0.1 μm) PBS.
  2. Incubate on ice or on a gentle shaker for 30 minutes at 4°C.
  3. Centrifuge again at the appropriate ultracentrifugation speed.
  4. Repeat the wash step once more.
  5. Following the final centrifugation, carefully remove all supernatant.
B. Fixation

Reagent

  • 2.5% Glutaraldehyde in 0.1 M sodium cacodylate buffer

Procedure

  1. Resuspend the EVP pellet in 2.5% glutaraldehyde solution.

    • The final volume depends on:
      • Starting sample volume
      • Expected particle yield
      • Number of TEM grids required

  2. As a guideline, prepare sufficient sample to apply 5 to 10 μL per TEM grid.

  3. Recommended EVP concentration:

    • 1 to 5 μg/μL for storage
    • Dilute as necessary before grid preparation

  4. Transfer the suspension into a clean 1.5 mL microcentrifuge tube.

  5. Store at 4°C until processing.

  6. Submit samples to FEMR for grid preparation and TEM imaging within 24 to 48 hours of fixation.

Important Notes

  • Avoid repeated freeze-thaw cycles.
  • Prolonged storage may alter vesicle morphology and increase aggregation.
  • Ensure fixation buffer is freshly prepared.
C. Negative Staining

Materials

  • Carbon-coated 200-mesh copper TEM grids
  • Pelco easiGlow™ discharge unit
  • Self-locking anti-capillary tweezers
  • Parafilm
  • Filter paper
  • 0.2 M glycine
  • Ultrapure water (ddH₂O)
  • 2% aqueous uranyl acetate (filtered immediately before use)

1. Grid Preparation

  1. Place carbon-coated TEM grids into the glow discharge holder with the carbon film facing upward.

  2. Glow discharge using a Pelco easiGlow:

    • 30 seconds
    • 30 mA

  3. Use grids within 20 minutes of glow discharge to ensure maximum surface hydrophilicity.

Quality Check

A properly glow-discharged grid should allow aqueous droplets to spread evenly across the carbon film without beading.

2. Sample Preparation

  1. Prepare at least two grids per specimen whenever possible.
  2. If the suspension is highly concentrated, dilute using ultrapure water.

Recommended Working Concentration

Approximately 0.05 to 0.1 μg/μL

⚠ Important: Phosphate-containing buffers may cause uranyl acetate precipitation and crystalline artifacts. If samples are stored in PBS, extensive water washing is required before staining.

3. Sample Application

Place clean parafilm on the glass plate in the negative staining workstation (Room SADB B/6).

Two sample adsorption methods may be used.

Method 1: Direct Application

Recommended for moderately concentrated samples.

  1. Hold the grid with self-locking tweezers.
  2. Apply 5 to 10 μL of sample directly onto the carbon surface.
  3. Incubate for 5 minutes.
  4. Remove excess liquid by gently touching the grid edge to filter paper.

Method 2: Drop Incubation

Recommended for dilute samples.

  1. Pipette 20 μL of sample onto parafilm.
  2. Float the grid on the sample drop with the carbon side facing downward.
  3. Incubate for 5 to 10 minutes.
  4. Cover the sample to minimize evaporation and contamination.

Increasing Particle Density

For dilute samples:

  1. Incubate for 10 minutes.
  2. Remove the grid.
  3. Transfer to a fresh sample drop.
  4. Repeat incubation one or more times as needed.

This approach often increases particle density and improves imaging efficiency.

4. Washing and Negative Staining

Glycine Quenching

Purpose: Neutralizes residual aldehyde fixative and reduces background staining.

  1. Transfer the grid (sample side down) sequentially across three drops of 0.2 M glycine.
  2. Incubate 2 minutes per drop.

Water Washing

Purpose: Remove salts and buffer components.

  1. Transfer the grid through five drops of ultrapure water.
  2. Incubate 1 to 2 minutes per drop.

Critical Step

Insufficient washing is one of the most common causes of:

  • Uranyl acetate precipitation
  • Crystalline deposits
  • Poor image contrast
  • Excessive background staining

Negative Staining

  1. Place the grid (sample side down) on a 20 μL drop of freshly filtered 2% uranyl acetate.
  2. Stain for 1 minute.
  3. Remove the grid and wick away excess stain from the edge using filter paper.

⚠ Do not touch the carbon film directly with the filter paper.

Alternative Stains

For acid-sensitive particles, consider:

  • Ammonium molybdate
  • Ammonium tungstate
  • Nano-W™
  • Uranyl formate

These stains may better preserve fragile particle morphology.

5. Drying and Storage

  1. Allow grids to air dry completely at room temperature for approximately 60 minutes.
  2. Alternatively, a low-heat lamp may be used to accelerate drying.
  3. Store dried grids in a clean TEM grid box.

Storage Recommendations

  • Best imaging quality: within several days of preparation.
  • Acceptable storage: 1 to 2 weeks in a dust-free container at room temperature.
  • Avoid high humidity environments.
TEM Imaging Recommendations

Initial Screening

  • Magnification: 10,000× to 30,000×
  • Assess particle density and stain quality.

Detailed Imaging

  • Magnification: 50,000× to 150,000×
  • Record representative fields from multiple grid regions.

Image Quality Indicators

A good negative stain preparation should exhibit:

✅ Even particle distribution
✅ Low background contamination
✅ Minimal salt crystals
✅ Good particle contrast
✅ Limited aggregation

Common Pitfalls and Troubleshooting

Poor Particle Adsorption

Possible causes

  • Insufficient glow discharge
  • Expired hydrophilic surface
  • Incorrect grid orientation

Solution

  • Verify carbon side orientation.
  • Use grids within 20 minutes of glow discharge.

Particle Aggregation

Possible causes

  • Over-concentrated sample
  • Excessive fixation
  • Prolonged storage

Solution

  • Dilute sample appropriately.
  • Prepare grids as soon as possible after fixation.

Salt or Crystal Artifacts

Possible causes

  • PBS contamination
  • Inadequate washing
  • Old stain solution

Solution

  • Increase water washes.
  • Filter stain immediately before use.

Low Particle Density

Possible causes

  • Dilute sample
  • Short adsorption time

Solution

  • Use repeated drop-incubation cycles.
  • Increase adsorption time to 10 minutes.

Poor Contrast

Possible causes

  • Insufficient staining time
  • Over-washing
  • Aged stain

Solution

  • Use freshly prepared stain.
  • Maintain consistent staining times.

Beam-Induced Damage

Possible causes

  • Incomplete drying
  • Prolonged exposure to a focused beam

Solution

  • Ensure grids are fully dry before imaging.
  • Use low-dose imaging procedures where appropriate.
Notes
  1. Extracellular vesicles frequently exhibit a "cup-shaped" morphology in negative stain TEM due to dehydration and drying artifacts and should not be interpreted as their native structure.
  2. Freshly prepared fixation and staining solutions generally provide the most consistent results.
  3. All incubation steps should be performed in a dust-free environment.
  4. When immunogold labeling is performed, all incubations should be carried out in a humidified chamber to prevent drying artifacts.
  5. Negative staining provides information on particle morphology and size distribution but does not preserve EVP structure as faithfully as cryo-electron microscopy (cryo-EM).
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