Gold Immunostaining of Exosomal Sections: A Technique to Visualize Diagnostic Markers in Exosomal Lumen
Published: April 30, 2023
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 Prepare 60 nm ultrathin sections of an exosome block using an ultra-microtome and collect them on a nickel grid. Incubate the grids in 50 µL drops of 0.02 M glycine for 10 min to quench the free aldehyde groups. Rinse in 100 μL of DW three times each for 10 min. Incubate at room temperature for 1 h in PBS containing 1% BSA. Incubate the grids in 50-100 µL drops of anti-KRS antibody (1:100 in PBS co…
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The authors have nothing to disclose.
Materials
| 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 |
