Fig. 1
From: A peptide tag-specific nanobody enables high-quality labeling for dSTORM imaging
Comparison and characterization of BC2-nanobody (BC2-Nb) formats for wide-field and dSTORM imaging. a Schematic illustration of the BC2-Nb dye-conjugation strategies. Monovalent and bivalent BC2-Nbs were either conjugated with Alexa Fluor 647 (AF647) via N-hydroxysuccinimide (NHS) ester (left panel) or linked to AF647 by enzymatic sortase coupling (right panel). Wide-field imaging of chemically fixed HeLa cells expressing mCherry-vimentinBC2T (mCherry-VIMBC2T) stained with modified BC2-Nbs. Monovalent versions of the BC2-Nbs (NHS- and sortase-coupled) are depicted on the left panel, corresponding bivBC2-Nbs are displayed on the right side. Stainings with NHS-conjugated nanobodies are shown in two different image contrasts, the upper half in the same brightness and contrast as the sortase-coupled nanobodies; in the lower half with an adjusted contrast. Scale bars, 25 µm. b Representative dSTORM images of chemically fixed HeLa cells expressing vimentinBC2T, stained with the monomeric NHS-conjugated BC2-NbAF647 (NHS) (left) and the sortase-coupled bivBC2-NbAF647 (sort) (right). Scale bars, images 5 µm, insets 1 µm. Image reconstruction details are given in Methods section. c Assessment of staining quality in wide-field fluorescence imaging. Labeling of the different nanobody formats was quantified by calculating the ratio of the signal intensity of mCherry-VIMBC2T expressing cells to non-transfected cells (background), (BC2-NbAF647 (NHS): n = 115; bivBC2-NbAF647 (NHS): n = 134; BC2-NbAF647 (sort): n = 150; bivBC2-NbAF647 (sort): n = 195) (Methods section, Supplementary Fig. 1). d Assessment of bivBC2-NbAF647 staining of endogenous β-catenin. Bar chart summarizes measured nanobody per µm2 values for untransfected chemically fixed HeLa cells stained with GFP-NbAF647 or bivBC2-NbAF647 in comparison to chemically fixed HeLa cells transiently expressing BC2TLC3B stained with bivBC2-NbAF647, errors given as standard deviation (S.D.), N = 3 cells for each condition (Methods section, Supplementary Fig. 2). e Quantification of completeness of labeling for the bivBC2-Nb and SNAP-tag labeling systems using FtnA-oligomers of 24 subunits. Bar chart summarizes median values of FtnA-24mer fluorescence intensities as percentage of theoretical maxima (Methods section, Supplementary Fig. 3)
