Gold Immunostaining of Exosomal Sections: A Technique to Visualize Diagnostic Markers in Exosomal Lumen

Abstract

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

In this video, we demonstrate gold immunostaining of biomarkers localized within the lumen region of ultrathin exosomal sections. This technique enables early diagnosis of diseases by visualization of target molecules within the exosomes secreted by the diseased cells.

Protocol

1. Immuno-staining of Exosome Sections and Imaging

1. Prepare 60 nm ultrathin sections of an exosome block using an ultra-microtome and collect them on a nickel grid.
2. Incubate the grids in 50 µL drops of 0.02 M glycine for 10 min to quench the free aldehyde groups.
3. Rinse in 100 μL of DW three times each for 10 min. Incubate at room temperature for 1 h in PBS containing 1% BSA.
4. Incubate the grids in 50-100 µL drops of anti-KRS antibody (1:100 in PBS containing 0.1% BSA) for 1 h (If necessary, this step should be carried out at 4 °C overnight.)
5. Wash the grids with five separate drops (50 µL) of PBS containing 0.1% BSA for 10 min each.
6. Transfer the grids to drops of secondary antibody for 1 h (anti-rabbit IgG conjugated to 10 nm gold particle (1:100) in PBS containing 0.1% BSA).
7. Wash the grids with five separate drops (50 µL) of PBS containing 0.1% BSA each 10 min.
8. Double-stain with 2% uranyl acetate for 20 min under dark conditions and with Reynold's lead citrate for 10 min, respectively.
9. Observe the grids under transmission electron microscopy at 80 kV.
10. Click "Acquire" and then click "File" and "Save as" in the CCD camera system under the TEM at 80 kV. Exposure time followed automatic settings.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Uranyl acetate	EMS	22400	Hazardous chemical
Lead citrate	EMS	17900
Transmission Electron Microscopy	Hitachi	H7600
Copper grid	EMS	G200-Cu
Glycine	SIGMA	022K5404
Phosphate buffer saline	SIGMA	P4417
Bovine serum albumin	Aurion	25557
Purified anti-human CD274 (B7- H1, PD-L1) antibody	BioLegend	329710	Whole mount Immumogold
Purified Mouse IgG2b, κ Isotype Ctrl	BioLegend	401202	Whole mount Immumogold (Control)
2nd Anti-mouse IgG conjugated 9-11 nm gold particle	sigma	G7652
Paraformaldehyde	EMS	19210