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Cited by 296Advanced Electron Microscopy Techniques and ApplicationsAdvanced Materials Characterization TechniquesMedical and Biological Sciences

High-contrast en bloc staining of neuronal tissue for field emission scanning electron microscopy

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Tapia, Juan Carlos, Kasthuri, Narayanan, Hayworth, Kenneth J, Schalek, Richard, Lichtman, Jeff W, Smith, Stephen J, Buchanan, JoAnn·2012·Nature Protocols
DOI 10.1038/nprot.2011.439PMC PMC3701260
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Abstract

Conventional heavy metal poststaining methods on thin sections lend contrast but often cause contamination. To avoid this problem, we tested several en bloc staining techniques to contrast tissue in serial sections mounted on solid substrates for examination by field emission scanning electron microscopy (FESEM). Because FESEM section imaging requires that specimens have higher contrast and greater electrical conductivity than transmission electron microscopy (TEM) samples, our technique uses osmium impregnation (OTO) to make the samples conductive while heavily staining membranes for segmentation studies. Combining this step with other classic heavy metal en bloc stains, including uranyl acetate (UA), lead aspartate, copper sulfate and lead citrate, produced clean, highly contrasted TEM and scanning electron microscopy (SEM) samples of insect, fish and mammalian nervous systems. This protocol takes 7-15 d to prepare resin-embedded tissue, cut sections and produce serial section images.

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  • Potassium ferrocyanide and potassium ferricyanide facilitate the reconstruction of cellular processes and the automatic segmentation of electron microscope images.

    “These compounds facilitate the reconstruction of cellular processes and the automatic segmentation of electron microscope images.”

    From Unambiguous identification of asymmetric and symmetric synapses using volume electron microscopy (2024) · cites this paper as Tapia et al., 2012

    The Membrane Contrasting Agents section says either potassium ferricyanide or ferrocyanide improves osmium staining of membranes. The Introduction links the staining method to segmentation of neuronal membranes and 3D reconstruction, but the copy does not establish that these compounds facilitate automatic image segmentation specifically. Page numbers are unavailable in our copy. (LLM, conf=0.82)

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  • Volume electron microscopy studies have commonly employed potassium ferrocyanide or potassium ferricyanide.

    “However, volume electron microscopy studies have commonly employed potassium ferrocyanide (Harris and Stevens, 1988, 1989; Harris et al., 1992, 2015; Medalla et al., 2007; Hua et al., 2015) or potassium ferricyanide (Tapia et al., 2012).”

    From Unambiguous identification of asymmetric and symmetric synapses using volume electron microscopy (2024) · cites this paper as Tapia et al., 2012

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Authors
Tapia, J. C., Kasthuri, N., Hayworth, K. J., Schalek, R., Lichtman, J. W., Smith, S. J., & Buchanan, J.
Affiliations
  • Harvard University (US)
  • Stanford Medicine (US)
Corresponding author
Buchanan, JoAnn
ORCID
Venue
Nature Protocols
Publisher
Springer Science and Business Media LLC
Published
2012-01-12
Volume
7
Issue
2
Pages
193-206
ISSN
  • 1750-2799 (ISSN-L)
  • 1754-2189
DOI
10.1038/nprot.2011.439
PMC
PMC3701260
OpenAlex
W2036100004
Cited by
296
References
3
Citations by year
2026: 9 · 2025: 24 · 2024: 24 · 2023: 26 · 2022: 23 · 2021: 33 · 2020: 26 · 2019: 27 · 2018: 26 · 2017: 16 · 2016: 20 · 2015: 20 · 2014: 7 · 2013: 9 · 2012: 6
FWCI
6.05
Type
journal article
Language
en
Open access
Licence
www.springer.com/tdm (tdm)
Locations
  • Nature Protocols (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • Howard Hughes Medical Institute
  • National Institute of Neurological Disorders and Stroke (R01NS075252)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Advanced Electron Microscopy Techniques and Applications
  • Physical Sciences › Engineering › Biomedical Engineering › Advanced Materials Characterization Techniques
  • Health Sciences › Medicine › Anatomy › Medical and Biological Sciences
Keywords
en bloc staining, neuronal tissue, field emission scanning electron microscopy, heavy metal staining, uranyl acetate, copper sulfate, lead citrate, electrical conductivity, membrane staining, insect nervous system, fish nervous system, mammalian nervous system, serial sections
MeSH terms
Animals, Aspartic Acid, Brain, Cell Membrane, Drosophila, Lead, Microscopy, Electron, Scanning, Microtomy, Nerve Tissue, Organometallic Compounds, Osmium, Staining and Labeling, Zebrafish, Copper Sulfate, Citric Acid, Mice

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