通过相关光电子显微镜成像VIPER标记的细胞蛋白
Imaging VIPER-labeled Cellular Proteins by Correlative Light and Electron Microscopy.
作者信息
Doh Julia K, Chang Young Hwan, Enns Caroline A, Lόpez Claudia S, Beatty Kimberly E
机构信息
Department of Biomedical Engineering, Oregon Health & Science University, Portland, Oregon 97239, USA.
OHSU Center for Spatial Systems Biomedicine, Oregon Health & Science University, Portland, Oregon 97239, USA.
出版信息
Bio Protoc. 2019 Nov 5;9(21):e3414. doi: 10.21769/BioProtoc.3414.
Advances in fluorescence microscopy (FM), electron microscopy (EM), and correlative light and EM (CLEM) offer unprecedented opportunities for studying diverse proteins and nanostructures involved in fundamental cell biology. It is now possible to visualize and quantify the spatial organization of cellular proteins and other macromolecules by FM, EM, and CLEM. However, tagging and tracking cellular proteins across size scales is restricted by the scarcity of methods for attaching appropriate reporter chemistries to target proteins. Namely, there are few genetic tags compatible with EM. To overcome these issues we developed Versatile Interacting Peptide (VIP) tags, genetically-encoded peptide tags that can be used to image proteins by fluorescence and EM. VIPER, a VIP tag, can be used to label cellular proteins with bright, photo-stable fluorophores for FM or electron-dense nanoparticles for EM. In this Bio-Protocol, we provide an instructional guide for implementing VIPER for imaging a cell-surface receptor by CLEM. This protocol is complemented by two other Bio-Protocols outlining the use of VIPER ( Doh , 2019a and 2019b).
荧光显微镜（FM）、电子显微镜（EM）以及相关光电子显微镜（CLEM）技术的进步为研究基础细胞生物学中涉及的多种蛋白质和纳米结构提供了前所未有的机遇。现在，通过FM、EM和CLEM技术可以对细胞蛋白质和其他大分子的空间组织进行可视化和定量分析。然而，跨尺寸尺度标记和追踪细胞蛋白质受到将合适的报告化学物质连接到目标蛋白质的方法稀缺的限制。也就是说，与EM兼容的遗传标签很少。为了克服这些问题，我们开发了通用相互作用肽（VIP）标签，这是一种基因编码的肽标签，可用于通过荧光和EM对蛋白质进行成像。VIPER作为一种VIP标签，可用于用明亮、光稳定的荧光团标记细胞蛋白质以用于FM，或用电子致密纳米颗粒标记以用于EM。在本生物协议中，我们提供了一份使用VIPER通过CLEM对细胞表面受体进行成像的操作指南。该协议由另外两份概述VIPER使用方法的生物协议（Doh，2019a和2019b）补充。
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