Jung Min Kyo, Mun Ji Young
Department of Convergence Medicine, University of Ulsan College of Medicine and Asan Institute for Life Sciences, Asan Medical Center.
Synaptic Circuit Plasticity Laboratory, Department of Structure and Function of Neural Network, Korea Brain Research Institute;
J Vis Exp. 2018 Jan 4(131):56482. doi: 10.3791/56482.
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' 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'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.
外泌体是由体液分泌的纳米级细胞外囊泡，已知其代表分泌它们的细胞的特征。分泌囊泡的内容物和形态反映了细胞行为或生理状态，例如细胞生长、迁移、分裂和死亡。外泌体的作用可能高度依赖于大小，外泌体的大小在30到300纳米之间变化。最广泛使用的外泌体成像方法是负染色，而其他结果基于冷冻透射电子显微镜、扫描电子显微镜和原子力显微镜。通过负染色评估的典型外泌体形态为杯状，但进一步细节尚不清楚。特别是在医学和制药领域，通过结构研究充分表征外泌体是必要的。因此，应通过电子显微镜技术（如在外泌体详细结构中标记特定蛋白质）来验证功能依赖的形态。为了观察详细结构，比较了外泌体的超薄切片图像和负染色图像。在本方案中，我们建议使用透射电子显微镜对外泌体进行成像，包括负染色、整装免疫染色、包埋制备、薄切片和免疫金标记。
