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Immunolabeling artifacts and the need for live-cell imaging.
Ulrike Schnell1, Freark Dijk, Klaas A Sjollema
1Department of Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
Nature Methods
|February 1, 2012
Summary
Live-cell imaging using fluorescent fusion proteins offers advantages over traditional immunofluorescence. Immunofluorescence can alter protein localization in fixed cells, making live-cell studies crucial for accurate protein localization research.
Area of Science:
- Cell biology
- Molecular imaging
Background:
- Fluorescent fusion proteins enable visualization of proteins in living cells.
- Immunofluorescence studies face criticism due to protein modification and ectopic expression.
- Immunoreagents can disrupt native protein localization in fixed cells.
Purpose of the Study:
- To highlight the limitations of immunofluorescence in protein localization studies.
- To advocate for the integration of live-cell imaging techniques.
- To ensure accurate in vivo protein localization analysis.
Main Methods:
- Discussion of potential pitfalls in immunofluorescence labeling.
- Comparison between fixed-cell immunostaining and live-cell imaging.
- Analysis of protein behavior in living versus permeabilized cells.
Main Results:
- Immunofluorescence can lead to protein extraction or relocalization.
- Fixed-cell immunostaining may not accurately represent in vivo protein localization.
- Live-cell imaging provides a more faithful representation of native protein behavior.
Conclusions:
- Immunofluorescence studies on dead, permeabilized cells have inherent limitations.
- Live-cell imaging is essential for validating protein localization data.
- Combining live-cell imaging with immunostaining ensures robust protein localization findings.
