Circumvention of common labelling artefacts using secondary nanobodies

2020 | journal article; research paper. A publication with affiliation to the University of Göttingen.

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​Circumvention of common labelling artefacts using secondary nanobodies​
Sograte-Idrissi, S.; Schlichthaerle, T.; Duque-Afonso, C. J. ; Alevra, M.; Strauss, S.; Moser, T.   & Jungmann, R. et al.​ (2020) 
Nanoscale12(18) pp. 10226​-10239​.​ DOI: https://doi.org/10.1039/d0nr00227e 

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Authors
Sograte-Idrissi, Shama; Schlichthaerle, Thomas; Duque-Afonso, Carlos J. ; Alevra, Mihai; Strauss, Sebastian; Moser, Tobias ; Jungmann, Ralf; Rizzoli, Silvio O. ; Opazo, Felipe 
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.
Issue Date
2020
Journal
Nanoscale 
Project
EXC 2067: Multiscale Bioimaging 
Working Group
RG Moser (Molecular Anatomy, Physiology and Pathology of Sound Encoding) 
RG Rizzoli (Quantitative Synaptology in Space and Time) 
External URL
https://mbexc.uni-goettingen.de/literature/publications/185
ISSN
2040-3364
eISSN
2040-3372
Language
English

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