透射电镜下体的样品制备与成像
Summary
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。
Abstract
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。
Introduction
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。
Protocol
1. 外切块的制备、切片、染色和成像 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。 在室温下用1毫升的0.1 米甲缓冲液去除固定剂并冲?…
Representative Results
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。 <img alt=…
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Glutaraldehyde | EMS | 16200 | |
| Sodium cacodylate | EMS | 12300 | |
| Osmium tetroxide 1 g crystal | EMS | 19100 | Use only in fume hood |
| acetone | JUNSEI | 1B2031 | |
| Spurr medium | TED PELLA | 18108 | |
| Ultra-microtome | Leica | UCT | |
| Uranyl acetate | EMS | 22400 | Hazardous chemical |
| Lead citrate | EMS | 17900 | |
| Transmission Electron Microscopy | Hitachi | H7600 | |
| nickel grid | EMS | G200-Ni | |
| Copper grid | EMS | G200-Cu | |
| glycine | SIGMA | 022K5404 | |
| Phosphate buffer saline | SIGMA | P4417 | |
| Bovine serum albumin | Aurion | 25557 | |
| 1st Antibody | purification in manually | ||
| Purified anti-human CD274 (B7-H1, PD-L1) Antibody | BioLegend | 329710 | Whole mount Immumogold |
| Purified Mouse IgG2b, κ Isotype Ctrl | BioLegend | 401202 | Whole mount Immumogold (Control) |
| 2nd Anti-mouse igG conjugated 9-11 nm gold particle | sigma | G7652 | |
| silver paint | CANS | CTP100 | |
| aluminum stub | EMS | 75622 | |
| FIB-SEM | Zeiss | AURIGA | |
| Transmission Electron Microscopy | JEOL | JEM2200FS | |
| Glow discharger | JEOL | JFC1100E | |
| Carbon grid | EMS | 121119 | |
| Paraformaldehyde | EMS | 19210 | |
| Formvar carbon coated Copper Grid (200 meh) | EMS | FCF200-CU | |
| Formvar carbon coated Nickel Grid (200 mesh) | EMS | FCF200-NI |
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Cite This Article
Jung, M. K., Mun, J. Y. Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy. J. Vis. Exp. (131), e56482, doi:10.3791/56482 (2018).