Circumvention of common labelling artefacts using secondary nanobodies

Sograte-Idrissi, Shama; Schlichthaerle, Thomas; Duque-Afonso, Carlos J.; Alevra, Mihai; Strauss, Sebastian; Moser, Tobias; Jungmann, Ralf; Rizzoli, Silvio O. und Opazo, Felipe (2020): Circumvention of common labelling artefacts using secondary nanobodies. In: Nanoscale, Bd. 12, Nr. 18: S. 10226-10239

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.

• Dokumententyp: Zeitschriftenartikel
• Fakultät: Physik
• Themengebiete: 500 Naturwissenschaften und Mathematik > 530 Physik
• ISSN: 2040-3364
• Sprache: Englisch