Format

Send to

Choose Destination
See comment in PubMed Commons below
Nat Methods. 2015 Jun;12(6):568-76. doi: 10.1038/nmeth.3365. Epub 2015 Apr 27.

High-performance probes for light and electron microscopy.

Author information

1
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
2
Department of Neurobiology &Anatomy, University of Utah, Salt Lake City, Utah, USA.
3
Howard Hughes Medical Institute, Department of Chemistry &Chemical Biology, Harvard University, Cambridge, Massachusetts, USA.
4
1] Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA. [2] Laboratory of Systems Neuroscience, National Institute of Mental Health, Bethesda, Maryland, USA.
5
Laboratory of Systems Neuroscience, National Institute of Mental Health, Bethesda, Maryland, USA.
6
1] Howard Hughes Medical Institute, Department of Chemistry &Chemical Biology, Harvard University, Cambridge, Massachusetts, USA. [2] Howard Hughes Medical Institute, Department of Physics, Harvard University, Cambridge, Massachusetts, USA.

Abstract

We describe an engineered family of highly antigenic molecules based on GFP-like fluorescent proteins. These molecules contain numerous copies of peptide epitopes and simultaneously bind IgG antibodies at each location. These 'spaghetti monster' fluorescent proteins (smFPs) distributed well in neurons, notably into small dendrites, spines and axons. smFP immunolabeling localized weakly expressed proteins not well resolved with traditional epitope tags. By varying epitope and scaffold, we generated a diverse family of mutually orthogonal antigens. In cultured neurons and mouse and fly brains, smFP probes allowed robust, orthogonal multicolor visualization of proteins, cell populations and neuropil. smFP variants complement existing tracers and greatly increase the number of simultaneous imaging channels, and they performed well in advanced preparations such as array tomography, super-resolution fluorescence imaging and electron microscopy. In living cells, the probes improved single-molecule image tracking and increased yield for RNA-seq. These probes facilitate new experiments in connectomics, transcriptomics and protein localization.

PMID:
25915120
PMCID:
PMC4573404
DOI:
10.1038/nmeth.3365
[Indexed for MEDLINE]
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Nature Publishing Group Icon for PubMed Central
    Loading ...
    Support Center