Issue 18, 2020

Circumvention of common labelling artefacts using secondary nanobodies


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.

Graphical abstract: Circumvention of common labelling artefacts using secondary nanobodies

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Article information

Article type
08 Jan 2020
17 Apr 2020
First published
24 Apr 2020
This article is Open Access
Creative Commons BY license

Nanoscale, 2020,12, 10226-10239

Circumvention of common labelling artefacts using secondary nanobodies

S. Sograte-Idrissi, T. Schlichthaerle, C. J. Duque-Afonso, M. Alevra, S. Strauss, T. Moser, R. Jungmann, S. O. Rizzoli and F. Opazo, Nanoscale, 2020, 12, 10226 DOI: 10.1039/D0NR00227E

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