Cited by 506Advanced Fluorescence Microscopy TechniquesCell Image Analysis TechniquesCellular transport and secretion

Immunolabeling artifacts and the need for live-cell imaging

Schnell, Ulrike, Dijk, Freark, Sjollema, Klaas A, Giepmans, Ben N G·2012·Nature Methods

Abstract

Fluorescent fusion proteins have revolutionized examination of proteins in living cells. Still, studies using these proteins are met with criticism because proteins are modified and ectopically expressed, in contrast to immunofluorescence studies. However, introducing immunoreagents inside cells can cause protein extraction or relocalization, not reflecting the in vivo situation. Here we discuss pitfalls of immunofluorescence labeling that often receive little attention and argue that immunostaining experiments in dead, permeabilized cells should be complemented with live-cell imaging when scrutinizing protein localization.

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Crossref · 10.1038/nmeth.1855

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  • Methanol fixation extracts lipids, dehydrates and permeabilizes cells, causing proteins to denature and precipitate, whereas paraformaldehyde fixation cross-links proteins and macromolecules in place.

    “Methanol fixation extracts lipids, dehydrates and permeablizes cells causing proteins to denature and precipitate onto the cellular architecture. In contrast, para-formaldehyde fixation cross-links proteins and other macromolecules in place 71.”

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Authors
Schnell, U., Dijk, F., Sjollema, K. A., & Giepmans, B. N. G.
Affiliations
University Medical Center Groningen (NL)
Corresponding author
Giepmans, Ben N G
Venue
Nature Methods
Published
2012-01-30
Volume
9
Issue
2
Pages
152-158
ISSN
  • 1548-7091 (ISSN-L)
  • 1548-7105
OpenAlex
W2048608623
Cited by
506
References
44
Citations by year
2026: 27 · 2025: 39 · 2024: 26 · 2023: 39 · 2022: 38 · 2021: 39 · 2020: 38 · 2019: 40 · 2018: 42 · 2017: 39 · 2016: 34 · 2015: 40 · 2014: 27 · 2013: 24 · 2012: 14
FWCI
15.34
Type
journal article
Language
en
Open access
closed
Locations
  • Nature Methods (publishedVersion)
  • PubMed (publishedVersion)
  • University of Groningen research database (University of Groningen / Centre for Information Technology) (submittedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Cell Image Analysis Techniques
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
Keywords
subcellular protein localization, live-cell imaging, fluorescent protein fusion, immunofluorescence, cell fixation, cell permeabilization, protein extraction, protein relocalization, ectopic expression
MeSH terms
Aldehydes, Cell Line, Cell Membrane Permeability, Cells, Fluorescent Antibody Technique, Humans, Proteins, Artifacts

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