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).
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Funding was provided by the US National Institutes of Health (R01-CA186568 to A.Y.T.; P41 GM103412 and R01GM086197 to M.H.E.) and a Howard Hughes Medical Institute Collaborative Initiative Award (to A.Y.T.). J.D.M. and S.S.L. were supported by National Science Foundation Graduate Research and National Defense Science and Engineering fellowships.
The Massachusetts Institute of Technology has submitted a patent application related to this work.
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Martell, J., Deerinck, T., Lam, S. et al. Electron microscopy using the genetically encoded APEX2 tag in cultured mammalian cells. Nat Protoc 12, 1792–1816 (2017). https://doi.org/10.1038/nprot.2017.065
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