Circumvention of common labelling artefacts using secondary nanobodies

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. The discovery and validation of nanobodies against specific targets are challenging, thus only a minimal amount of them are currently available. Here, using STED, DNA-PAINT, and light-sheet microscopy, we 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 artificial probe-induced clustering of target molecule seen with conventional 2.Abs in living or poorly fixed samples. Altogether, showing that secondary nanobodies are a valuable alternative to 2.Abs.