Anchoring and labeling of proteins using acrydite-modified oligonucleotides (oligos) in the original ExM protocol. a Chemical structure of the acrydite-modified oligo. b Schematic of microtubules (green) and hydrogel network (orange). c The acrydite-modified oligo (shown in a) hybridized to a oligo-bearing secondary antibody (gray, top), bound via the primary antibody (gray, bottom) to the microtubules (purple). The acrydite-oligo is incorporated into the gel (orange lines) via the methacryloyl group (orange dot) and remains anchored after the removal of the microtubules and antibodies by proteolysis (dotted lines). d Pre-expansion widefield fluorescence image of Thy1-YFP mouse brain slice. Scale bar, 500 μm. e Post-expansion widefield image of the same sample in d. Scale bar, 500 μm (2.01 mm). f, g Confocal fluorescence image of the boxed regions in d and e, respectively. Scale bars, 5 μm (20.1 μm) h, i Confocal fluorescence image of the boxed regions in f and g, respectively. Scale bars, 2.5 μm (10.0 μm).