Circumvention of common labelling artefacts using secondary nanobodies

Nanoscale. 2020 May 14;12(18):10226-10239. doi: 10.1039/d0nr00227e.


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.

MeSH terms

  • Animals
  • Artifacts*
  • COS Cells
  • Chlorocebus aethiops
  • Cochlea / innervation
  • Cochlea / pathology
  • DNA / chemistry
  • Fluorescent Dyes / chemistry
  • Immunoassay / methods*
  • Mice
  • Microscopy, Confocal
  • Microtubules / immunology
  • Single-Domain Antibodies / chemistry
  • Single-Domain Antibodies / immunology*


  • Fluorescent Dyes
  • Single-Domain Antibodies
  • DNA