Protocol | Published:

Electron microscopy using the genetically encoded APEX2 tag in cultured mammalian cells

Nature Protocols volume 12, pages 17921816 (2017) | Download Citation

Abstract

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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Acknowledgements

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.

Author information

Affiliations

  1. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Jeffrey D Martell
    • , Stephanie S Lam
    •  & Alice Y Ting
  2. National Center for Microscopy and Imaging Research, University of California at San Diego, La Jolla, California, USA.

    • Thomas J Deerinck
    •  & Mark H Ellisman
  3. Department of Neurosciences, University of California at San Diego, La Jolla, California, USA.

    • Mark H Ellisman
  4. Department of Bioengineering, University of California at San Diego, La Jolla, California, USA.

    • Mark H Ellisman
  5. Department of Genetics, Stanford University, Stanford, California, USA.

    • Alice Y Ting
  6. Department of Biology, Stanford University, Stanford, California, USA.

    • Alice Y Ting
  7. Department of Chemistry, Stanford University, Stanford, California, USA.

    • Alice Y Ting

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Contributions

J.D.M. and A.Y.T. developed the original APEX tag for electron microscopy. S.S.L. and A.Y.T. developed APEX2. T.J.D. and M.H.E. developed protocols for cell staining, EM sample processing, and imaging by light and electron microscopy. J.D.M. prepared all constructs and cell samples for the figures, and T.J.D. performed all EM sample processing and imaging. J.D.M. wrote the paper. All authors edited the paper.

Competing interests

The Massachusetts Institute of Technology has submitted a patent application related to this work.

Corresponding authors

Correspondence to Mark H Ellisman or Alice Y Ting.

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DOI

https://doi.org/10.1038/nprot.2017.065

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