Adapted and Prepared by Jeannie Mui
Negative Staining and Immunogold Labelling of Extracellular Vesicles and Particles (EVPs)
Ultracentrifugation protocol (from Thermo Fisher Scientific)
- 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 °C), pour off supernatant and add conditioned media until the entire volume is processed into a pellet.
- 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.
- Resuspend the pellet in 2.5% glutaraldehyde fixative solution in 0.1M sodium cacodylate buffer (final pellet resuspension depends on how much the starting material is, 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.
- Transfer EVPs to a 1.5 mL Eppendorf test tube and store them at 4 °C.
- Bring to the FEMR for negative staining and TEM imaging within one to two days.
(1) Optimized Protocol for Negative Staining of Extracellular Vesicles and Particles (EVPs)
(2) Negative Staining and Immunogold Labelling of Extracellular Vesicles and Particles (EVPs)
(1) 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
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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.
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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 a 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.
(2) Negative Staining and Immunogold Labelling of Extracellular Vesicles and Particles (EVPs)
Materials
- Purified EVPs or exosomes
- 4% Paraformaldehyde (PFA) in 0.1 M phosphate buffer
- 200-mesh copper TEM grids with carbon film
- PBS, DPBS, ddH₂O
- 0.2 M Glycine solution
- 1% BSA in PBS
- Primary antibody (e.g., anti-PD-L1)
- Secondary antibody conjugated to gold particles (e.g., 10 nm; dilution 1:20)
- 1% Glutaraldehyde in 0.1 M sodium cacodylate buffer
- 2% Uranyl acetate
- Parafilm
- Glow discharge unit
- Humid chamber
Protocol
1. Fixation
- Mix purified EVPs 1:1 with 4% PFA in 0.1 M phosphate buffer.
- Gently resuspend and incubate at room temperature.
- Proceed to staining within 1–2 hours.
2. Grid Preparation
- Glow discharge TEM grids (carbon side up) for 30 seconds at 20 µA.
- Place a clean sheet of Parafilm on the glass plate at the negative staining station (FEMR, Room B/5).
3. Sample Application
- Apply 5–7 µL of EVP solution to the carbon side of the grid.
- Incubate for 15 minutes at room temperature.
4. Washing
- Float the grid (sample side down) on three successive drops of 100 µL PBS, 5 minutes each.
- Repeat with three drops of 50 µL 0.2 M glycine, 3 minutes each (to quench free aldehydes).
5. Blocking
- Float the grid on a drop of PBS containing 1% BSA for 5 minutes.
6. Primary Antibody Incubation
- Incubate grid with 20 µL of primary antibody diluted in PBS with 0.1% BSA.
- Incubate for 1 hour at room temperature or overnight at 4°C in a humid chamber.
7. Washing
- Wash grid on five drops of DPBS, 5 minutes each.
8. Secondary Antibody Incubation
- Block again with 1% BSA in PBS for 5 minutes.
- Incubate with 20 µL of gold-conjugated secondary antibody (1:20 dilution in 0.1% BSA) for 1 hour at room temperature.
9. Final Washing
- Wash grid on five drops of DPBS, 5 minutes each.
- Wash on five drops of ddH₂O, 2 minutes each.
10. Post-Fixation
- Float grid on a drop of 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 2 minutes.
- Wash on five drops of ddH₂O, 1 minute each.
11. Negative Staining
- Stain with 2% uranyl acetate for 1 minute.
- Blot excess stain and air dry or under a heat lamp.
12. Imaging
- Image grids using transmission electron microscopy (TEM).
- Alternatively, store grids in a labelled TEM grid box for future analysis.
Notes
- Exosomes typically appear cup-shaped due to dehydration and vacuum artifacts during TEM preparation.
- Ensure all antibody incubations are performed in a humidified environment to prevent drying.
- Use freshly prepared staining and fixation solutions for optimal results.