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. 2021 Jul 6;220(9):e202105067. doi: 10.1083/jcb.202105067

Figure 1.

Figure 1.

Workflow and characterization of LR-ExM.(A) Workflow of LR-ExM. (B) Schematic of trifunctional anchors. (C–E) ExM confocal images of CCPs in U2OS cells indirectly immunostained for clathrin heavy-chain (POI). (C) ProExM using AF488-conjugated secondary antibodies. (D) ExM with postexpansion labeling using biotin-conjugated antibodies. (E) LR-ExM using antibodies conjugated with NHS-MA-biotin trifunctional anchor. Samples in D and E were postexpansion stained with streptavidin-AF488. (F) Intensity quantification of C–E. Error bars represent SD. n = 3 for each case. (G) LR-ExM confocal image of CCPs in U2OS cells immunostained indirectly with secondary antibodies conjugated with NHS-MA-DIG anchor, postexpansion stained with anti-DIG antibody. (H and I) Cross sections of the CCP in the boxed area of G. The length expansion ratios for images in C, D,E, and G–I are 4.3, 4.5, 4.6, and 4.3, respectively. The length expansion ratio for the samples used in plot F is 4.5 ± 0.2. Scale bars, 500 nm (C–E and G) and 100 nm (H and I). All scale bars are in preexpansion units. arb. u., arbitrary units; STV, streptavidin.