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

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.