This article discusses the visualization of exosomal biomarkers using electron microscopy techniques. It outlines the steps involved in preparing exosomal sections and the staining process to enhance contrast for better observation.
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.
To begin, incubate grids containing 60-nanometer thick unstained sections in 50-microliter drops of 0.02 M glycine for 10 minutes to quench the free aldehyde groups. Then, rinse the sections in 100 microliters of distilled water three times for 10 minutes each. Following the last rinse, incubate the sections for 1 hour at room temperature in PBS containing 1% BSA. Then, incubate the grids in 50- to 100-microliter drops of an anti-KRS antibody for 1 hour.
Next, wash the grids five times for 10 minutes each in a drop of PBS containing 0.1% BSA. Then, transfer the grids to a drop of an appropriate secondary antibody and incubate the sections for 1 hour at room temperature. Now, wash the grids five times for 10 minutes each with a separate drop of PBS containing 0.1% BSA. Double stain the sections with 2% uranyl acetate for 20 minutes in the dark, followed by Reynold's lead citrate for 10 minutes. Place the stained section into a transmission electron microscope and view the sample using 80 kV and image them using the microscope software.
繁體中文
