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Journal Article

Circumvention of common labelling artefacts using secondary nanobodies

MPS-Authors
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Schlichthaerle,  Thomas
Jungmann, Ralf / Molecular Imaging and Bionanotechnology, Max Planck Institute of Biochemistry, Max Planck Society;

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Strauss,  Sebastian
Jungmann, Ralf / Molecular Imaging and Bionanotechnology, Max Planck Institute of Biochemistry, Max Planck Society;

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Jungmann,  Ralf
Jungmann, Ralf / Molecular Imaging and Bionanotechnology, Max Planck Institute of Biochemistry, Max Planck Society;

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Fulltext (public)

d0nr00227e.pdf
(Publisher version), 5MB

Supplementary Material (public)

d0nr00227e1.pdf
(Supplementary material), 2MB

Citation

Sograte-Idrissi, S., Schlichthaerle, T., Duque-Afonso, C. J., Alevra, M., Strauss, S., Moser, T., et al. (2020). Circumvention of common labelling artefacts using secondary nanobodies. Nanoscale, 12(18), 10226-10239. doi:10.1039/d0nr00227e.


Cite as: http://hdl.handle.net/21.11116/0000-0006-934B-0
Abstract
A standard procedure to study cellular elements is via immunostaining followed by optical imaging. This methodology typically requires target-specific primary antibodies (1.Abs), which are revealed by secondary antibodies (2.Abs). Unfortunately, the antibody bivalency, polyclonality, and large size can result in a series of artifacts. Alternatively, small, monovalent probes, such as single-domain antibodies (nanobodies) have been suggested to minimize these limitations. The discovery and validation of nanobodies against specific targets are challenging, thus only a minimal amount of them are currently available. Here, we used STED, DNA-PAINT, and light-sheet microscopy, to demonstrate that secondary nanobodies (1) increase localization accuracy compared to 2.Abs; (2) allow direct pre-mixing with 1.Abs before staining, reducing experimental time, and enabling the use of multiple 1.Abs from the same species; (3) penetrate thick tissues more efficiently; and (4) avoid probe-induced clustering of target molecules observed with conventional 2.Abs in living or poorly fixed samples. Altogether, we show how secondary nanobodies are a valuable alternative to 2.Abs.