Sectioning, Staining and TEM Imaging of Embedded Exosomes: A Protocol To Visualize the Structural Features of Exosomes Using TEM

Samenvatting

Source: Jung, M. K. et al. Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy. J. Vis. Exp. (2018)

This video describes the technique for preparing a thin section of embedded exosomes. These sections can further be stained and imaged using transmission electron microscopy, or TEM, to study the structural features of exosomes.

Protocol

1. Sectioning of the Exosomes

1. Prepare exosome block by embedding the purified exosome sample (obtained from the culture supernatant of HCT116 cells) in pure low viscosity embedding mixture.
2. Section the exosome block with 60 nm thickness through an ultra-microtome.
3. Double stain with 2% uranyl acetate for 20 min and lead citrate for 10 min.
   For Reynolds lead solution, add 1.33 g of lead nitrate and 1.76 g of sodium citrate to a total of 50 mL distilled water.
4. Observe the grid under transmission electron microscopy at 80 kV.
5. Click "Acquire" and then click "File" and "Save as" in the CCD camera system under the electron microscope at 80 kV. Follow automatic settings for the exposure time.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Ultra-microtome	Leica	UCT
Uranyl acetate	EMS	22400	Hazardous chemical
Lead citrate	EMS	17900
Transmission Electron Microscopy	Hitachi	H7600
Nickel grid	EMS	G200-Ni
Copper grid	EMS	G200-Cu
Transmission Electron Microscopy	JEOL	JEM2200FS