Circumvention of common labeling artifacts using secondary nanobodies

Abstract

The most common procedure to reveal the location of specific (sub)cellular elements in biological samples is via immunostaining followed by optical imaging. This is typically performed with target-specific primary antibodies (1.Abs), which are revealed by fluorophore-conjugated secondary antibodies (2.Abs). However, at high resolution this methodology can induce a series of artifacts due to the large size of antibodies, their bivalency, and their polyclonality. Here we use STED and DNA-PAINT super-resolution microscopy or light sheet microscopy on cleared tissue to show how monovalent secondary reagents based on camelid single-domain antibodies (nanobodies; 2.Nbs) attenuate these artifacts. We demonstrate that monovalent 2.Nbs have four additional advantages: 1) they increase localization accuracy with respect to 2.Abs; 2) they 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) they penetrate thick tissues efficiently; and 4) they avoid the artificial clustering seen with 2.Abs both in live and in poorly fixed samples. Altogether, this suggests that 2.Nbs are a valuable alternative to 2.Abs, especially when super-resolution imaging or staining of thick tissue samples are involved.

Colocalization of different neurotransmitter transporters on synaptic vesicles is sparse except for VGLUT1 and ZnT3

“…Copy number measurement Isolated SVs from rat brain were labeled either for VGLUT1 or for ZnT3 using primary monoclonal antibody and secondary nanobody (Sograte-Idrissi et al, 2020) conjugated to a definite number of fluorophores resulting in a fixed stoichiometry between the number of protein molecules and fluorophores. Following single vesicle imaging, the copy number was obtained by dividing the SV fluorescence signal with the mean signal of the free antibody-nanobody complex (Figures 4E and 4F).…”

“…N1202). Two fluorophores (Star RED, Abberior) are site-specifically coupled to one nanobody molecule ensuring a fixed stoichiometry of 4 to 1 (two nanobodies bind to each F(ab) of IgG molecule) for the number of fluorophores per IgG (Shaw et al, 2019;Sograte-Idrissi et al, 2020). Initially, 2.5fold excess of secondary nanobody was added to the monoclonal primary antibody to saturate available binding sites of IgG.…”

Expansion microscopy: A powerful nanoscale imaging tool for neuroscientists

“…While increasing the expansion factor by improving the gel chemistry can lead to higher resolution, reducing the size of labels may also be helpful, as small labels have reduced biomolecule localization error: the farther away a fluorophore is from the biomolecule, the less precisely its location can be determined ( Gao et al, 2019a ; Shi et al, 2019 ; Zwettler et al, 2020 ). Multiple groups have been exploring small labels with size much smaller than antibody, for example, nanobodies ( Sograte-Idrissi et al, 2019 ) and small genetically encoded tags ( Shi et al, 2019 ). Commercially available antibody fragments may be another excellent option.…”