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

Exosomes are small extracellular vesicles enclosing a variety of biomolecules that serve as cancer biomarkers.

To visualize exosomal biomarkers, begin with ultrathin resin-embedded exosomal sections supported on suitable electron microscopy metal grids. Incubate the grids in glycine drops. Glycine binds and neutralizes reactive aldehyde groups in the sections to overcome their interference with the assay.

Rinse the sample with water to remove residual reagents. Subsequently, incubate the sample in a blocking buffer. The buffer carries serum proteins that block non-specific antibody binding sites in the sample.

Next, incubate the sample in the desired primary antibody solution. These antibodies recognize and bind to their target biomarkers localized in the exosomal lumen. Wash off any unbound antibodies with a suitable buffer.

Now, transfer the sample-bearing grids to drops containing gold-conjugated secondary antibodies that attach to the primary antibodies. The gold-nanoparticles serve as electron-dense markers to visualize the fine localization of target molecules. Wash off any unbound secondary antibodies with a suitable buffer.

Finally, under dark conditions, incubate the grids sequentially in uranyl acetate and lead citrate solutions. These heavy metal reagents stain and enhance the contrast of organic molecules like lipids, proteins, and glycogens in the sample, differentiating them from the background.

Observe the stained sections under a transmission electron microscope. Gold-labeled biomarkers appear as dark particles within the lumen of heavy metal-stained exosome structures.