Encyclopedia of Experiments: Cancer Research
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Negative Staining of Purified Exosomes - ExperimentTranscript
To perform negative staining of exosomes - nanosized, spherical vesicles - begin by taking a suitable electron microscopy grid. Prep the grid using a glow-discharger. This process makes the grid surface hydrophilic for better adhesion of samples.
Simultaneously, take a suspension of purified exosomes in a tube. Supplement the tube with paraformaldehyde - a sample fixative - and incubate. Paraformaldehyde penetrates the exosomes and cross-links the proteins and nucleic acids. This step is vital for preserving the morphology of exosomes.
Now, load the exosome suspension onto the pre-prepared grid and incubate to allow adsorption to occur. Next, add a few drops of uranyl acetate, a heavy metal stain, over the grid. Uranyl ions bind to the membrane proteins and lipids, forming a deposit around the exosome boundary.
Last, wash the grid in water to remove the excess stain. Allow the grid to dry and image using Transmission Electron Microscopy or TEM.
When an electron beam strikes the exosomes, the stain surrounding the specimen scatters more electrons. Exosomes being less electron-dense, allow the beam to pass through. These differently scattered electrons are captured by the imaging system to form a high contrast image.
Negatively stained exosomes appear lighter, with a dark boundary around them.
