Immunolabeling artifacts and the need for live-cell imaging
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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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.”
From Xeroderma Pigmentosa Group A (XPA), Nucleotide Excision Repair and Regulation by ATR in Response to Ultraviolet Irradiation (2017) · cites this paper as [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
- Licence
- www.springer.com/tdm (tdm)
- 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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