Enhanced dSTORM Immunostaining Protocol

To achieve the higher labeling density required for dSTORM imaging the staining protocol was slightly modified. Cells were prepared the same way as described up to the storage step in PBS. After storage, cells were blocked with 10% (w/V) BSA (Carl Roth, cat. #8076) in PBS for 30 min, then additionally with Image-iT FX signal enhancer (ThermoFisher Scientific, cat. #I36933) for 60 min. Antibodies and nanobodies were diluted to ~0.5 µg ml⁻¹ in staining/permeabilization solution (PBS, 10% BSA, 0.1% (V/V) Triton X-100 (Sigma-Aldrich, cat. #T8787)). Conventional immunostaining was done at 4 °C for 24 h with the primary antibody (V9, mouse monoclonal, Sigma-Aldrich, cat. #347M-1)^{72 (link)} followed by two washes with PBS, and then stained at 4 °C for 24 h with the secondary antibody (donkey-anti-mouse AF647, ThermoFisher Scientific, cat. #A-31571). For staining with nanobodies, cells were incubated at 4 °C for 48 h. Unbound Nbs were removed by two washes with PBS-T (0.1% w/V Tween-20 (Sigma-Aldrich, cat. #P7949)) and samples were post-fixed with 4% PFA (Sigma-Aldrich, cat. #F8775) and 0.25% (w/V) glutaraldehyde (Sigma-Aldrich, cat. #G5882) in PBS for 5 min to make the binding permanent. Finally, cells were washed twice with PBS to remove fixation solution and stored in PBS with 0.1% (w/V) sodium azide (Carl Roth, cat. #4221) until imaging.