This research was supported by the Bio &#38; Medical Technology Development Program of the National Research Foundation (NRF) &#38; funded by the Korean government (MSIP&#38;MOHW) (No. 2016M3A9B6904244). We also thank members of the Medicinal Bioconvergence Research Center for purification of exosome.

1. Block Preparation, Sectioning, Staining and Imaging of the Exosome
<ol>
	<li>Pellet the exosomes from culture supernatant of HCT116 cells by centrifugation at 100,000 x g for 1.5 h23. Remove the culture supernatant and carefully fix the purified exosome pellet with 1 mL of 2.5% glutaraldehyde in 0.1 M sodium cacodylate solution (pH 7.0) for 1 h at 4 &#176;C. For 0.1 M sodium cacodylate, dissolve 4.28 g cacodylic acid in 160 mL of distilled water (DW). Adjust pH to 7.4 with 0.1 M HCl then make...

<ol>
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(49), (2011).</li><li>Wang, J., Yao, Y., Wu, J., Li, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+analysis+of+exosomes+secreted+from+macrophages+extracted+by+different+methods.">Identification and analysis of exosomes secreted from macrophages extracted by different methods.</a> Int J Clin Exp Pathol. 8 (6), 6135-6142 (2015).</li><li>Thery, C., Amigorena, S., Raposo, G., Clayton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+exosomes+from+cell+culture+supernatants+and+biological+fluids.">Isolation and characterization of exosomes from cell culture supernatants and biological fluids.</a> Curr Protoc Cell Biol. , 22 (2006).</li><li>Mazzeo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosome+secretion+by+eosinophils:+A+possible+role+in+asthma+pathogenesis.">Exosome secretion by eosinophils: A possible role in asthma pathogenesis.</a> J Allergy Clin Immunol. 135 (6), 1603-1613 (2015).</li><li>Sokolova, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+exosomes+derived+from+human+cells+by+nanoparticle+tracking+analysis+and+scanning+electron+microscopy.">Characterisation of exosomes derived from human cells by nanoparticle tracking analysis and scanning electron microscopy.</a> Colloids Surf B Biointerfaces. 87 (1), 146-150 (2011).</li><li>Sharma, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural-mechanical+characterization+of+nanoparticle+exosomes+in+human+saliva,+using+correlative+AFM,+FESEM,+and+force+spectroscopy.">Structural-mechanical characterization of nanoparticle exosomes in human saliva, using correlative AFM, FESEM, and force spectroscopy.</a> ACS Nano. 4 (4), 1921-1926 (2010).</li><li>Raposo, G., Stoorvogel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles:+exosomes,+microvesicles,+and+friends.">Extracellular vesicles: exosomes, microvesicles, and friends.</a> J Cell Biol. 200 (4), 373-383 (2013).</li><li>Bosch, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trehalose+prevents+aggregation+of+exosomes+and+cryodamage.">Trehalose prevents aggregation of exosomes and cryodamage.</a> Sci Rep. 6, 36162 (2016).</li><li>Lobb, R. 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This article presents a protocol for observing the detailed exosome&#39;s structure and labeling of its specific proteins. Negative staining has been considered as the best method for exosome imaging17. This conventional technique has shown exosomes&#39; cup-shaped structure. However, this cup shape is a form of artefact that can occur due to the drying process. Cryo-TEM results have shown that exosomes have a perfectly spherical structure in aqueous solution25,<...

Extracellular vesicles (EVs) are lipid bilayer vesicles secreted by cells, and their size ranges between 30 and 300 nm. EVs were first reported in 1978, with evidence from vesicles of patients with Hodgkin&#39;s disease1. These vesicles were reported to be 40 to 120 nanometers in size. In 1980, it was reported that EVs are involved in the coagulation system and thrombosis, formation of a blood clot inside a blood vessel in cancer patients2. After 30 years, EVs have been reported to be an important factor to promote tumor invasion, immune escape, and angiogenesis3. In addition, the functions of EVs have been studied as regulators in cellular interactions in the areas of inflammation, immune disorder, neurological disease, and cancer4. Since EVs contain specific biomolecules such as protein, mRNA, and microRNA5, their potential application in diagnostics and therapeutics has been analyzed6,7. EVs are a category composed of subgroups including exosomes, prostasomes, oncosomes, dexosomes, microparticles, promininosomes, argosomes, and exosome-like vesicles, depending on their cellular origin and biological function3. In addition, based on their biogenesis, these EVs can be divided into microvesicles (shed microvesicles, 100 - 1000 nm) and exosomes (30 - 300 nm)8,9. Among these, the exosome has been reported as a cell communicator for immune response10, cancer11,12, and infectious disease13.
Interest in the exosomes as a biomarker for early diagnosis is increasing rapidly, and the purification and characterization of exosomes must be accompanied with molecular imaging techniques. The varying sizes and morphologies of exosomes, depending on their origin and function14, can be distinguished by microscopy techniques with high resolution, such as electron microscopy. Most exosomes were visualized by negative stained Transmission Electron Microscopy (TEM)15,16,17, and these results were confirmed by immunolabeling of a specific protein in whole mount vesicle18. Several research groups have reported the structure through Scanning Electron Microscopy, Atomic Force Microscopy19,20, and Cryo-TEM21,22. However, while these techniques are useful for studying the exosome structure, they are insufficient for observing the position of specific proteins located inside the exosome. Therefore, we introduced a protocol for imaging exosomes with a specific protein&#39;s labelling. We applied block preparation, ultrathin sectioning, and immunostaining for ascertaining the protein&#39;s detailed location in the exosome. This was compared with negative staining and whole mount immunostaining, which is traditionally used for characterization of the exosome.

<table border="0" cellpadding="0" cellspacing="0" width="583">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Glutaraldehyde</td>
			<td style="width:81px;">EMS</td>
			<td style="width:119px;">16200</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Sodium cacodylate&#160;</td>
			<td>EMS</td>
			<td style="width:119px;">12300</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Osmium tetroxide 1 g crystal</td>
			<td>EMS</td>
			<td style="width:119px;">19100</td>
			<td>Use only in fume hood</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">acetone</td>
			<td>JUNSEI</td>
			<td>1B2031</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Spurr medium</td>
			<td>TED PELLA</td>
			<td style="width:119px;">18108</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Ultra-microtome</td>
			<td style="width:81px;">Leica</td>
			<td style="width:119px;">UCT</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Uranyl acetate</td>
			<td>EMS</td>
			<td style="width:119px;">22400</td>
			<td>Hazardous chemical</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Lead citrate</td>
			<td>EMS</td>
			<td style="width:119px;">17900</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Transmission Electron Microscopy</td>
			<td style="width:81px;">Hitachi</td>
			<td style="width:119px;">H7600</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">nickel grid</td>
			<td style="width:81px;">EMS</td>
			<td style="width:119px;">G200-Ni</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Copper grid</td>
			<td>EMS</td>
			<td style="width:119px;">G200-Cu</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">glycine</td>
			<td>SIGMA</td>
			<td>022K5404</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Phosphate buffer saline</td>
			<td style="width:81px;">SIGMA</td>
			<td style="width:119px;">P4417</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Bovine serum albumin</td>
			<td style="width:81px;">Aurion</td>
			<td style="width:119px;">25557</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">1st Antibody</td>
			<td style="width:81px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">purification in manually&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Purified anti-human CD274 (B7-H1, PD-L1) Antibody</td>
			<td style="width:81px;">BioLegend</td>
			<td style="width:119px;">329710</td>
			<td style="width:167px;">Whole mount Immumogold</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Purified Mouse IgG2b, &#954; Isotype Ctrl</td>
			<td style="width:81px;">BioLegend</td>
			<td style="width:119px;">401202</td>
			<td style="width:167px;">Whole mount Immumogold (Control)</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">2nd Anti-mouse igG conjugated 9-11 nm gold particle</td>
			<td style="width:81px;">sigma</td>
			<td style="width:119px;">G7652</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">silver paint</td>
			<td style="width:81px;">CANS</td>
			<td style="width:119px;">CTP100</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">aluminum stub</td>
			<td style="width:81px;">EMS</td>
			<td style="width:119px;">75622</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">FIB-SEM</td>
			<td style="width:81px;">Zeiss</td>
			<td style="width:119px;">AURIGA</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Transmission Electron Microscopy</td>
			<td style="width:81px;">JEOL</td>
			<td style="width:119px;">JEM2200FS</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Glow discharger</td>
			<td style="width:81px;">JEOL</td>
			<td style="width:119px;">JFC1100E</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Carbon grid</td>
			<td>EMS</td>
			<td style="width:119px;">121119</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Paraformaldehyde</td>
			<td>EMS</td>
			<td style="width:119px;">19210</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Formvar carbon coated Copper Grid (200 meh)</td>
			<td style="width:81px;">EMS</td>
			<td style="width:119px;">FCF200-CU</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Formvar carbon coated&#160; Nickel Grid (200 mesh)</td>
			<td style="width:81px;">EMS</td>
			<td style="width:119px;">FCF200-NI</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

sample preparation and imaging of exosomes by transmission electron microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

Currently, exosomes are classified into size and shape categories by transmission electron microscopy. Figure 2 shows negative stained exosome and immune-labelled exosome in whole mount status. Figure 3 shows sectioned exosome and immuno-labelled exosomes after thin sectioning. Immuno-gold staining using antibodies of specific proteins is used to positively identify an exosome and classify the types of proteins in the exosome. Th...

Watch this Scientific Journal Video about Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy at JoVE.com

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

The overall goal of this procedure is to observe the morphology of purified extracellular vesicles and to perform specific protein localization within extracellular vesicles using electron microscopy. This method can help answer key questions in the extracellular vesicle research period, such as the categorization of the exosome and specific proteins located inside of it. The main advantage of this technique is that it can be used to observe the portion of specific proteins located both inside and outside the exosome.The implication of this technique is to ensure diagnosis of several disease, including cancer and bacteria infections. Though this method can provide insight into the exosome, it can also be applied to other biological samples, microcellular incubation and specific protein localization. Generally, individuals new to this method will struggle because of create contamination while non-specific binding of immunogold particle.Using standard techniques, prepare exosomes in culture. Then ultracentrifuge the culture supernatant of HCT116 cells to pellet the exosomes from the media. Once pelleted, carefully remove the culture supernatant.To fix the purified exosome pellet, add one milliliter of 2.5%glutaraldehyde in a 0.1 molar soldium cacodylate solution at PH 7.0. Incubate the lipid bylayer vesicles for one hour at four degrees celsius. Next, remove the fixative and rinse the pellets with one milliliter of a 0.1 molar sodium cacodylate buffer solution at room temperature for 10 minutes.Then postfix the samples with one milliliter of 2%osmium tetroxide for one hour at four degrees celsius. Remove the fixative and rinse the exosome pellet three times with the 0.1 molar sodium cacodylate buffer, changing the buffer every ten minutes. Then dehydrate the sample by shaking it for 10 minutes each in a series of graded acetone concentrations.Next, mix up a solution of three parts acetone and one part of low viscosity embedding mixture. Replace the acetone with this mixture and incubate the sample for 30 minutes. Continue to increase the ratio of the low viscosity embedding medium, incubating for 30 minutes with each step.Finally, incubate the exosome pellet in 100%of the low viscosity embedding mixture overnight at room temperature. The following day, embed the sample in pure low viscosity embedding mixture using an embedding mold and bake it for 24 hours at 65 degrees celsius. Using an ultramicrotome, prepare sections with a thickness of 60 nanometers.Place the sections on a nickel grid and double stain some of them with 2%uranyl acetate for 20 minutes, followed by lead citrate for 10 minutes. Next, place the stained section into a transmission electron microscope and view the sample using 80 kilovolts and follow automatic settings for the exposure time. With an exosome image in view, acquire and save the image using the microscope software.To begin, incubate grids containing 60 nanometer thick, unstained sections in 50 microliter drops of 0.02 molar 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 one 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 one 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 one 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 reynolds lead citrate for 10 minutes. Place the stained section into a transmission electron microscope and view the sample using 80 kilovolts and image them as previously shown.In order to perform negative staining, fix the purified exosomes following isolation with one milliliter of 2%paraformaldehyde for five minutes. Treat thin, formvar/carbon film coated, 200 mesh, copper EM grids with glow discharge for one minute, then load five to seven microliters of the exosome suspension solution on a grid and incubate it for one 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 grids 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. Start by treating the thin formvar/carbon film coated, 200 mesh copper EM grids with glow discharge for 30 seconds. Then, load five to seven microliters of the exosomes fixed in 2%paraformaldehyde solution on the grid and incubate them there for five minutes.Next, rinse the exosomes with 100 microliters of PBS, three times each for 10 minutes. Treat the exosome containing grids with 50 microliters of a 0.5 molar glycine solution for 10 minutes to quench the free aldehyde groups. Then, transfer the grids to a drop of PBS containing 1%BSA and block for 30 minutes.Now incubate the grids with 50 to 100 microliters of anti-PD-L1 antibody for one hour at room temperature or overnight at four degrees celsius. Wash the grid with five separate drops of PBS containing 0.1%BSA for 10 minutes each, then transfer the grid to a drop of the secondary antibody for one hour. Next, wash the grid with five separate drops of PBS containing 0.1%BSA for 10 minutes each, then wash the grid with two separate drops of distilled water.When finished, perform negative staining with 2%uranyl acetate, as previously shown, and image the samples, or store them in an EM grid box for future observation. The exosome morphology observed here is the product of negative staining. These exosomes show a typical cup shaped morphology, which is an artifact that can occur during the drying process.Additional information can be gained using whole mount immunogold staining, such as the location of specific proteins in the exosome. Here, the white arrows indicate the location of PD-L1. In sectioned images, the vesicles showed a lumen structure that is known as a structural feature of exosomes.These too can be immunogold stained to identify specific proteins throughout the exosomes. Once mastered, the immunogold staining technique can be done in fibrous cells following simple preparation and sectioning if it is performed properly. Following this procedure, other method like double immuno staining can be performed to simultaneously identify the location of two different proteins.After each development, this technique may be used in the field of extracellular vesicles to explore disease diagnoses in biopsies. After watching this video, you should have a good understanding of how to identify specific protein localization within the exosome. Don't forget they're working with fixative solutions, such as chlorhydrate, paraformaldehyde, and osmium tetroxide can be extremely hazordous and precautions such as ventilated fume hood and latex gloves should always be taken while performing this procedure.

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37.8K Views.  Asan Medical Center. The overall goal of this procedure is to observe the morphology of purified extracellular vesicles and to perform specific protein localization within extracellular vesicles using electron microscopy. This method can help answer key questions in the extracellular vesicle research period, such as the categorization of the exosome and specific proteins located inside of it. The main advantage of this technique is that it can be used to observe the portion of specific proteins located both insid...