Name: sample preparation and imaging of exosomes by transmission electron microscopy
Uploaded: 2018-01-04T13:00:00
Description: Watch this Scientific Journal Video about Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy at JoVE.com

这项研究得到了生物和 #38 的支持; 国家研究基金会 (NRF) 和 #38 的医疗技术发展方案; 由韩国政府资助 (MSIP 和 #38; 保健) (No. 2016M3A9B6904244)。我们还感谢医学 Bioconvergence 研究中心的成员外切纯化。

1. 外切块的制备、切片、染色和成像
<ol>
	<li>将 HCT116 细胞培养上清液中的体, 在 10万 x g 为 1.5 h23进行离心。去除培养上清液, 在1° c 的 0.1 M 钠甲溶液 (pH 7.0) 中, 用1毫升2.5% 戊二醛仔细修复纯化的外切颗粒, 4 小时。对0.1 米甲钠, 溶解4.28 克胂酸在160毫升蒸馏水 (DW)。将 pH 值调整到7.4 与0.1 米 HCl, 然后用蒸馏水构成200毫升。</li>
	<li>在室温下用1毫升的0.1 米甲缓冲液去除固定剂并冲?...

<ol>
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本文提出了一个协议, 以观察详细的外切的结构和标签的具体蛋白质。阴性染色被认为是外切成像17的最佳方法。这种传统的技术已经显示体的杯状结构。然而, 这个杯子形状是一种形式的人造制品, 可能发生由于干燥过程。低温透射电镜结果表明, 体在水溶液中具有完美的球形结构25,26。低温透射电镜技术是检测天然结构的一种非常有?...

胞外囊泡 (EVs) 是由细胞分泌的脂质双层囊泡, 其大小范围介于30和 300 nm 之间。EVs 是第一次报告在 1978年, 与证据从囊性霍奇金病患者的水泡1。据报道, 这些小泡的大小是40到120纳米。据报道, 在 1980年, EVs 参与了凝血系统和血栓形成, 在癌症患者的血管内产生了血块2。30年后, EVs 被报道是促进肿瘤侵袭、免疫逃逸和血管生成的重要因素3。此外, 在炎症、免疫紊乱、神经系统疾病和癌症等领域的细胞相互作用中, EVs 的功能已被研究为调节因子4。由于 EVs 包含特定的生物分子, 如蛋白质, mRNA, 和 rna5, 它们在诊断和治疗方面的潜在应用已被分析6,7。EVs 是一个由子群组成的类别, 包括体、prostasomes、oncosomes、dexosomes、微粒、promininosomes、argosomes 和外切样泡, 这取决于它们的细胞来源和生物学功能3。此外, 根据它们的生物, 这些 EVs 可分为微 (棚微、100-1000 nm) 和体 (30-300 nm)8,9。其中, 外切被报告为免疫应答的细胞通信器10、癌症11、12和传染性疾病13。
对体作为早期诊断的生物标记物的兴趣正在迅速增加, 体的纯化和鉴定必须伴有分子成像技术。不同的大小和形态的体, 根据它们的起源和功能14, 可以区分高分辨率的显微镜技术, 如电子显微镜。大多数体是由负染色透射电镜 (TEM)15,16,17, 这些结果是通过 immunolabeling 的一个特定的蛋白在整个芒泡的确认18. 有几个研究小组通过扫描电子显微镜、原子力显微镜、19、20、低温-TEM21、22来报告该结构。然而, 虽然这些技术是有用的研究外切结构, 他们是不足够的观察位置的特定蛋白质位于外切。因此, 我们介绍了一个协议的成像体与特定的蛋白质的标签。我们采用了块制备, 超薄切片和免疫, 以确定蛋白质的详细位置在外切。这是与阴性染色和整个芒免疫, 这是传统用于表征的外切。

<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

体是一种由体液分泌的纳米细胞胞外囊, 它能代表分泌它们的细胞的特征。分泌囊泡的含量和形态反映细胞的行为或生理状态, 例如细胞生长、迁移、分裂和死亡。体的角色可能高度依赖于大小, 体的大小从30到 300 nm 不等。最广泛使用的方法外切成像是阴性染色, 而其他结果是基于低温透射电镜, 扫描电镜, 和原子力显微镜。典型的外切的形态通过阴性染色评估是一个杯子形状, 但进一步细节还不清楚。通过结构研究的外切良好在医学和药学领域是必要的。因此, 功能相关的形态学应该通过电子显微镜技术来验证, 例如在外切的详细结构中标注特定的蛋白质。对体的结构、超薄切片图像和负染色图像进行了对比观察。在本协议中, 我们建议传输电子显微镜的成像体包括阴性染色, 全贴剂免疫染色, 块准备, 薄切片, 免疫金标签。

目前, 体被分为大小和形状类别的透射电子显微镜。图 2显示了负染色外切和免疫标签外切在整个安装状态。图 3显示了切片后的分段外切和免疫标记体。免疫金染色使用特异蛋白抗体, 用于积极识别外切和分类的蛋白质类型的外切。本协议采用全贴免疫和免疫塑料分段外切。
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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

透射电镜下体的样品制备与成像

本协议描述了传输电子显微镜所需的各种技术, 包括阴性染色, 超薄切片的详细结构, 和免疫金标签, 以确定特定的蛋白质在体的位置。

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

Research

JoVE Journal

Biology

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

电子显微镜对胚胎的制备果蝇胚胎心管

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

科研

生物学

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

单病毒原子力显微镜和超分辨率荧光成像的样品制备

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam

低温电子显微镜样品制备借助于聚焦离子束

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

串联高压冷冻和植物组织中快速冷冻替代透射电子显微镜

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, ...

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

该做什么和不该做什么低温电子显微镜的：入门样品制备和高质量的数据采集为大分子三维重建

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state ...

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

准备厚生物样品的观察扫描透射电子断层扫描

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...

Miniaturized Sample Preparation for Transmission Electron Microscopy

透射电镜的小型化样品制备方法

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

高压冷冻、微波辅助对比度增强和最小树脂嵌入在体积成像中的生物样品制备

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...