<p class='jove_title'><strong>1. Negative Staining</strong></p>

<ol>
	<li>Glow-discharge the thin formvar/carbon film-coated 200 mesh copper EM grids for 1 min by glow discharger.</li>
	<li>Fix purified exosomes with 1 mL of 2% paraformaldehyde (PFA) for 5 min.<br />
	<strong>NOTE</strong>: Paraformaldehyde fumes are toxic. All work should be done in a ventilated fume hood.</li>
	<li value='3'>Load 5-7 &#181;L exosome suspension solution on the grid and incubate for 1 min. If the concentration of exosome is too high, dilute the concentration to one-half to one-fifth.</li>
	<li>Immediately stain with the ~20 drops of filtered 1% uranyl acetate (UA) solution on the surface of the EM grid by syringe.</li>
	<li>Remove the excess UA solution on the grid by contacting the grid edge with filter paper.</li>
	<li>Quickly rinse the grid with a drop of water. This step will remove the excess staining solution.</li>
	<li>Place the grid on the table by holding it with tweezers, and cover the grid partially with a culture dish to dry for 10 min under room temperature.</li>
	<li>Store the grid in an EM grid box for future observation by a TEM at 80 kV.</li>
</ol>

<table><tbody><tr><td>Uranyl acetate</td><td>EMS</td><td>22400</td><td>Hazardous chemical</td></tr><tr><td>Transmission Electron Microscopy</td><td>JEOL</td><td>JEM2200FS</td><td></td></tr><tr><td>Glow discharger</td><td>JEOL</td><td>JFC1100E</td><td></td></tr><tr><td>Paraformaldehyde</td><td>EMS</td><td>19210</td><td></td></tr><tr><td>Formvar carbon-coated Copper&lt;br/&gt; Grid (200 mesh)</td><td>EMS</td><td>FCF200-CU</td><td></td></tr><tr><td>Formvar carbon-coated Nickel&lt;br/&gt; Grid (200 mesh)</td><td>EMS</td><td>FCF200-NI</td><td></td></tr></tbody></table>

negative staining of purified exosomes: a procedure to visualize exosome morphology using transmission electron microscopy

Negative Staining of Purified Exosomes: A Procedure to Visualize Exosome Morphology Using Transmission Electron Microscopy

<p class='jove_content'>Source: Jung, M. K. <em>et al.</em> <a href='https://www.jove.com/video/56482' target='_blank'>Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy.</a> <em>J. Vis. Exp.</em> (2018)</p>
<p class='jove_content'>This video describes a procedure for negative staining of purified exosomes. The negatively stained exosomes are imaged using transmission electron microscopy. This technique helps visualize the morphology of exosomes.</p>

Source: Jung, M. K. et al. Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy. J. Vis. Exp. (2018)
This video describes a ...

<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>Last, wash the grid in water to remove the excess stain. Allow the grid to dry and image using Transmission Electron Microscopy or TEM.</p>
<p>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 sys</p>

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5.3K Views. 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 ...

Abstract

Protocol

Disclosures

<table><tbody><tr><td>Uranyl acetate</td><td>EMS</td><td>22400</td><td>Hazardous chemical</td></tr><tr><td>Transmission Electron Microscopy</td><td>JEOL</td><td>JEM2200FS</td><td></td></tr><tr><td>Glow discharger</td><td>JEOL</td><td>JFC1100E</td><td></td></tr><tr><td>Paraformaldehyde</td><td>EMS</td><td>19210</td><td></td></tr><tr><td>Formvar carbon-coated Copper<br/> Grid (200 mesh)</td><td>EMS</td><td>FCF200-CU</td><td></td></tr><tr><td>Formvar carbon-coated Nickel<br/> Grid (200 mesh)</td><td>EMS</td><td>FCF200-NI</td><td></td></tr></tbody></table>

Negative Staining of Purified Exosomes: A Procedure to Visualize Exosome Morphology Using Transmission Electron Microscopy

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Abstract

Protocol

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Materials

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Name	Company	Catalog Number	Comments
Uranyl acetate	EMS	22400	Hazardous chemical
Transmission Electron Microscopy	JEOL	JEM2200FS
Glow discharger	JEOL	JFC1100E
Paraformaldehyde	EMS	19210
Formvar carbon-coated Copper Grid (200 mesh)	EMS	FCF200-CU
Formvar carbon-coated Nickel Grid (200 mesh)	EMS	FCF200-NI