Negative Staining and Immunogold Labelling of Extracellular Vesicles and Particles (EVPs)
听
Materials
- Purified EVPs or exosomes
- 4% Paraformaldehyde (PFA) in 0.1 M phosphate buffer
- 200-mesh copper TEM grids with carbon film
- PBS, DPBS, ddH鈧侽
- 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)
- 2.5% 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鈥�2 hours.
2. Grid Preparation
- Glow discharge TEM grids (carbon side up) for 30 seconds at 20 碌A.
- Place a clean sheet of Parafilm on the glass plate at the negative staining station (FEMR, Room B/5).
3. Sample Application
- Apply 5鈥�7 碌L 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 碌L PBS, 5 minutes each.
- Repeat with three drops of 50 碌L 0.2 M glycine, each for 3 minutes (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 the grid with 20 碌L of primary antibody diluted in PBS with 0.1% BSA.
- Incubate for 1 hour at room temperature or overnight at 4掳C in a humid chamber.
7. Washing
- Wash the grid on five drops of DPBS (or PBS) for 5 minutes each.
8. Secondary Antibody Incubation
- Block again with 1% BSA in PBS for 5 minutes.
- Incubate with 20 碌L of gold-conjugated secondary antibody (1:20 dilution in 0.1% BSA) for 1 hour at room temperature.
9. Final Washing
- Wash the grid with five drops of DPBS (or PBS) for 5 minutes each.
- Wash with five drops of ddH鈧侽 for 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 ddH鈧侽 for 2 minute each.
11. Negative Staining
- Stain with 2% uranyl acetate for 1 minute.
- Blot excess stain and air-dry or place 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.