Circumvention of common labeling artifacts using secondary nanobodies

Shama Sograte-Idrissi, Thomas Schlichthaerle, Carlos J. Duque-Afonso, Mihai Alevra, Sebastian Strauss, Tobias Moser, Ralf Jungmann, Silvio Rizzoli, Felipe Opazo

Preprint posted on 25 October 2019

A novel, small, and smart tool in fluorescence microscopy: nanobodies.

Selected by Mariana De Niz

Categories: cell biology, immunology


The most common procedure to reveal the location of specific subcellular elements in biological samples is via immunostaining followed by optical imaging. Standard immunodetection approaches use typically a primary antibody which binds the protein of interest  and a secondary antibody that binds to the primary antibody and carries a detection element which can be either a fluorophore or a single strand of DNA (used in DNA-PAINT). Although this classical immunostaining procedure is widely used, important limitations are size (leading to displacement errors incompatible with super-resolution microscopy) and the ability of secondary antibodies to bind more than one epitope. An alternative to secondary antibodies that addresses these limitations are monovalent recombinant secondary nanobodies. Moreover, nanobodies are more suitable for staining thick biological samples, greatly reducing the incubation times usually needed when using secondary antibodies. Finally, nanobodies overcome the issues faced when performing classical multiplexed immunostaining, which requires primary antibodies to be raised in different species. Nanobodies can be pre-mixed with primary antibodies prior to staining, circumventing the species limitation. In this preprint, Sograte-Idrissi et al (1) explore the use and advantages of secondary nanobodies over antibodies.


Key findings and developments

General findings

  • In their work, the authors explored the use of secondary nanobodies for several microscopy applications.
  • They confirmed that usage of secondary nanobodies decreases linkage error in STED microscopy and DNA-PAINT and increases localization accuracy compared to secondary antibodies.
  • They showed that pre-mixing of secondary nanobodies with primary antibodies before staining is successful- leading to reduced experimental time, and allowing the use of multiple primary antibodies from the same animal species.
  • They demonstrate that secondary nanobodies are better able to penetrate thick tissues by using them in optically cleared samples,
  • They show that secondary nanobodies avoid artificial clustering sometimes observed when using secondary antibodies.
Figure 1. Advantages of secondary nanobody use. a) Nanobody use increases localisation accuracy. b) Pre-mixing of primary Ab and secondary Nb allows use of primary antibodies from the same animal species. c) Use of nanobodies avoids artificial clustering.

Specific findings

Secondary nanobodies provide higher staining accuracy than secondary antibodies

  • Using STED microscopy, the authors imaged COS-7 cells stained with a monoclonal primary anti-tubulin antibody directly conjugated to Abberior Star635P and further recognized by either a polyclonal secondary antibody or a monovalent secondary nanobody carrying AbberiorStar580.
  • An autocorrelation analysis was performed on these images to evaluate the staining accuracy of the secondary probes. Analysis of the secondary nanobody showed greater accuracy. This was confirmed upon staining peroxisomes within primary hippocampal neurons.
  • The authors then explored secondary nanobody accuracy in the context of DNA-PAINT. The microtubule network of a fibroblast cell line was stained with monoclonal primary antibodies against alpha-tubulin, and detected with a secondary nanobody coupled to a docking strand. Greater accuracy on the apparent diameter of microtubule filaments was achieved.

Bypassing the primary antibody animal species limitations

  • Pre-mixing the primary and secondary antibodies prior to incubating them with the sample would save experimental time and costs, however, this is currently not possible due to the polyclonality and the bivalency of secondary antibodies that result in aggregation of the primary-secondary antibody complex and failure to stain the intended target in the sample.
  • Bypassing the pre-mixing limitation with monovalent secondary probes would allow using several primary antibodies raised in the same species.
  • In their work, the authors used monoclonal antibodies raised in mice against different components (tubulin, Golgi and nuclear pore complex), and pre-mixed each with secondary nanobodies anti-mouse carrying different fluorophores. Upon staining COS7 cells, minimal background and negligible cross-talk was observed between the channels.
  • Using Exchange-PAINT they obtained a super-resolved view of a single neuronal synapse in 3D using two primary antibodies from the same species.

Secondary nanobodies enhance sample penetration in shorter incubation time

  • The authors pre-mixed primary antibodies with secondary nanobodies for use in a complex thick sample: cochleae extracted from mice. They then compared how long the primary/secondary antibody and the primary antibody/secondary nanobody combinations need, to achieve homogeneous staining of the sample. They then imaged the sample after decalcification and optical clearing.
  • They observed faster homogeneous staining upon use of the primary antibody-secondary nanobody mix.

Secondary nanobodies reduce probe-induced clusters of target proteins on living cells

  • To test if the 2-nanobodies reduce probe-induced clustering of target molecules, the authors analysed the surface distribution of IgM containing B cell receptors (IgM-BCRs) on a human B cell line.
  • Cells were stained and chemically fixed with aldehydes to be imaged under stimulation emission depletion (STED) microscopy.
  • They found that the use of secondary nanobodies reduces probe-induced clusters, still observed using primary/secondary antibody combinations.

Probe-induced clusters of target proteins in aldehyde-fixed cells

  • Conventional fixations times with 4% PFA do not necessarily prevent protein movement. A more efficient fixative such as glutaraldehyde (GLU) could be used, but it generates unwanted autofluorescence and only few affinity molecules find their target epitopes after GLU crosslinking. An alternative is glyoxal, although its implementation is very recent.
  • Using primary/secondary antibody mixes, artefactual clustering formation was observed at most times of incubation with 4% PFA, except 30 minute incubation. Conversely, using secondary nanobodies had no significant change between live, 10 or 30 minutes of fixation with 4%PFA.


What I like about this preprint

I like that the authors explored and validated a relatively novel tool. Often, introducing new technologies to research can be a challenge, and thorough validation helps doing this.


Open questions

  1. You explored in great detail the comparison to secondary antibodies. Are there any limitations the science community should be aware of upon using nanobodies?
  2. Why in your opinion, is it still not a widely used tool?
  3. Are there any advantages in using nanobodies beyond fluorescence microscopy? Namely in other assays that also depend on immune complex formation?
  4. What are the characteristics of nanobodies in the context of photobleaching and blead-through?



  1. Sograte-Idrissi S, et al., Circumvention of common labelling artefacts using secondary nanobodies, 2019, bioRxiv,doi:10.1101/818351.


Posted on: 3 April 2020


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