Electron microscopy (EM) is the premiere technique for high-resolution imaging of cellular ultrastructure. Unambiguous identification of specific proteins or cellular compartments in electron micrographs, however, remains challenging because of difficulties in delivering electron-dense contrast agents to specific subcellular targets within intact cells. We recently reported enhanced ascorbate peroxidase 2 (APEX2) as a broadly applicable genetic tag that generates EM contrast on a specific protein or subcellular compartment of interest. This protocol provides guidelines for designing and validating APEX2 fusion constructs, along with detailed instructions for cell culture, transfection, fixation, heavy-metal staining, embedding in resin, and EM imaging. Although this protocol focuses on EM in cultured mammalian cells, APEX2 is applicable to many cell types and contexts, including intact tissues and organisms, and is useful for numerous applications beyond EM, including live-cell proteomic mapping. This protocol, which describes procedures for sample preparation from cell monolayers and cell pellets, can be completed in 10 d, including time for APEX2 fusion construct validation, cell growth, and solidification of embedding resins. Notably, the only additional steps required relative to a standard EM sample preparation are cell transfection and a 2- to 45-min staining period with 3,3-diaminobenzidine (DAB) and hydrogen peroxide (H₂O₂).

中文翻译：

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在培养的哺乳动物细胞中使用遗传编码的APEX2标签进行电子显微镜检查。

电子显微镜（EM）是细胞超微结构高分辨率成像的首要技术。然而，由于难以将电子致密的造影剂递送至完整细胞内的特定亚细胞靶标，因此在电子显微照片中明确鉴定特定蛋白质或细胞区室仍然具有挑战性。我们最近报道了增强的抗坏血酸过氧化物酶2（APEX2）作为广泛应用的遗传标签，可以在特定蛋白质或感兴趣的亚细胞区隔上产生EM对比。该协议提供了设计和验证APEX2融合构建体的指南，以及有关细胞培养，转染，固定，重金属染色，嵌入树脂和EM成像的详细说明。尽管此协议的重点是培养的哺乳动物细胞中的EM，APEX2适用于多种细胞类型和环境，包括完整的组织和生物体，可用于EM以外的众多应用，包括活细胞蛋白质组图谱分析。该协议描述了从细胞单层和细胞沉淀制备样品的程序，可以在10天内完成，包括APEX2融合构建体验证，细胞生长和包埋树脂固化的时间。值得注意的是，相对于标准EM样品制备，所需的唯一其他步骤是细胞转染以及使用3,3-二氨基联苯胺（DAB）和过氧化氢（H 其中描述了从细胞单层和细胞沉淀制备样品的程序，可以在10天内完成，包括APEX2融合构建体验证，细胞生长和包埋树脂固化的时间。值得注意的是，相对于标准EM样品制备，所需的唯一其他步骤是细胞转染以及使用3,3-二氨基联苯胺（DAB）和过氧化氢（H 其中描述了从细胞单层和细胞沉淀制备样品的程序，可以在10天内完成，包括APEX2融合构建体验证，细胞生长和包埋树脂固化的时间。值得注意的是，相对于标准EM样品制备，所需的唯一其他步骤是细胞转染以及使用3,3-二氨基联苯胺（DAB）和过氧化氢（H₂ O ₂）。