PT  - JOURNAL ARTICLE
AU  - Shama Sograte-Idrissi
AU  - Thomas Schlichthaerle
AU  - Carlos J. Duque-Afonso
AU  - Mihai Alevra
AU  - Sebastian Strauss
AU  - Tobias Moser
AU  - Ralf Jungmann
AU  - Silvio Rizzoli
AU  - Felipe Opazo
TI  - Circumvention of common labeling artifacts using secondary nanobodies
AID  - 10.1101/818351
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 818351
4099  - http://biorxiv.org/content/early/2019/10/25/818351.short
4100  - http://biorxiv.org/content/early/2019/10/25/818351.full
AB  - 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.