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1. Negative Staining

<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. 
	NOTE: 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 concentr...

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

Source: Jung, M. K. et al. <a href="https://www.jove.com/video/56482" target="_blank">Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy.</a> J. Vis. Exp. (2018)
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.

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

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

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.

negative-staining-purified-exosomes-procedure-to-visualize-exosome

Research

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Cancer Research

Gastrointestinal Cancer

Assays and techniques

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3 . Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high&#8208;resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5. Moreover, OpNS can be a high&#8208;throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.

More than half of proteins are small proteins (molecular mass &#60;200 kDa) that are challenging for both electron microscope imaging and three-dimensional reconstructions. Optimized negative staining is a robust and high-throughput protocol to obtain high contrast and relatively high resolution (~1 nm) images of small asymmetric proteins or complexes under different physiological conditions.

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of ...

JoVE Journal

Environment

Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural biology 1. In recent years, this method has achieved great success in studying structures of proteins and macromolecular complexes 2, 3. Compared with electron cryomicroscopy (cryoEM), in which frozen hydrated protein samples are embedded in a thin layer of vitreous ice 4, negative staining is a simpler sample preparation method in which protein samples are embedded in a thin layer of dried heavy metal salt to increase specimen contrast 5. The enhanced contrast of negative stain EM allows examination of relatively small biological samples. In addition to determining three-dimensional (3D) structure of purified proteins or protein complexes 6, this method can be used for much broader purposes. For example, negative stain EM can be easily used to visualize purified protein samples, obtaining information such as homogeneity/heterogeneity of the sample, formation of protein complexes or large assemblies, or simply to evaluate the quality of a protein preparation.
In this video article, we present a complete protocol for using an EM to observe negatively stained protein sample, from preparing carbon coated grids for negative stain EM to acquiring images of negatively stained sample in an electron microscope operated at 120kV accelerating voltage. These protocols have been used in our laboratory routinely and can be easily followed by novice users.

Visualizing protein samples by negative stain electron microscopy (EM) has become a popular structural analysis method. It is useful for quantitative structural analysis, such as calculating a 3D reconstruction of the molecules being studied, and also for qualitative examination of the quality of protein preparations. In this article we present detailed protocols for preparing the EM grids, staining the sample and visualizing the sample in an electron microscope. Novice users can follow these protocols easily and to utilize negative stain EM as a routine assay, in addition to other biochemical assays, for evaluating their protein samples.

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural ...

Bioengineering

Variations on Negative Stain Electron Microscopy Methods: Tools for Tackling Challenging Systems

Negative stain electron microscopy (EM) allows relatively simple and quick observation of macromolecules and macromolecular complexes through the use of contrast enhancing stain reagent. Although limited in resolution to a maximum of ~18 - 20 &#197;, negative stain EM is useful for a variety of biological problems and also provides a rapid means of assessing samples for cryo-electron microscopy (cryo-EM). The negative stain workflow is straightforward method; the sample is adsorbed onto a substrate, then a stain is applied, blotted, and dried to produce a thin layer of electron dense stain in which the particles are embedded. Individual samples can, however, behave in markedly different ways under varying staining conditions. This has led to the development of a large variety of substrate preparation techniques, negative staining reagents, and grid washing and blotting techniques. Determining the most appropriate technique for each individual sample must be done on a case-by-case basis and a microscopist must have access to a variety of different techniques to achieve the highest-quality negative stain results. Detailed protocols for two different substrate preparation methods and three different blotting techniques are provided, and an example of a sample that shows markedly different results depending on the method used is shown. In addition, the preparation of some common negative staining reagents, and two novel Lanthanide-based stains, is described with discussion regarding the use of each.

Negative stain EM is a powerful technique for visualizing macromolecular structure, but different staining techniques can produce varying results in a sample dependent manner. Here several negative staining approaches are described in detail to provide an initial workflow for tackling the visualization of challenging systems.

Negative stain electron microscopy (EM) allows relatively simple and quick observation of macromolecules and macromolecular complexes through the use of ...

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

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