This article details the procedure for negative staining of exosomes, which are nanosized vesicles crucial for various biological processes. The method enhances imaging quality by providing contrast in electron microscopy.
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.
In order to perform negative staining, fix the purified exosomes following isolation with 1 milliliter of 2% paraformaldehyde for 5 minutes. Treat thin formvar/carbon film coated 200 mesh copper EM grids with glow discharge for 1 minute. Then, load 5 to 7 microliters of the exosome suspension solution on a grid and incubate it for 1 minute. If the concentration of exosome is too high, dilute the concentration to an appropriate level.
Immediately, stain the exosomes with about 20 drops of a filtered 1% uranyl acetate solution on the surface of the EM grid. Remove the excess uranyl acetate solution from the grid by contacting the grid's edge with filter paper. Then, quickly rinse the grid with a drop of water.
Use a pair of tweezers to place the grid on the table, and cover the grid partially with a culture dish. Allow the grid to dry for 10 minutes at room temperature and then image the grid or store it in an electron microscope grid box for future observation.
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