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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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Figure 1: Fixation and permeabilization can affect epitope accessibility.
Figure 2: Effects of standard fixation and permeabilization protocols on protein localization and epitope accessibility in different cell lines.
Figure 3: Effects of standard immunostaining methods on protein extraction and EGFP fluorescence.
Figure 4: Ultrastructural changes after fixation and permeabilization.

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Acknowledgements

We thank H. van der Want and D. Hoekstra for critically reading the manuscript, A. Algra and R. Hoffmann for technical assistance, O. Sibon for H2B cDNA, H. Kampinga for expression constructs44 used in Figure 2c (University Medical Center Groningen) and V. Cirulli (University of Washington) for EpCAM cDNA. Part of this work was supported by the Groningen University Graduate School of Medical Sciences; a Marie Curie International Reintegration Grant within the 7th European Community Framework Program to B.N.G.G. and was performed at the University Medical Center Groningen Microscopy and Imaging Center, which is sponsored by Netherlands Organization for Scientific Research grants 40-00506-98-9021 and 175-010-2009-023.

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Correspondence to Ben N G Giepmans.

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Schnell, U., Dijk, F., Sjollema, K. et al. Immunolabeling artifacts and the need for live-cell imaging. Nat Methods 9, 152–158 (2012). https://doi.org/10.1038/nmeth.1855

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