Alternative land-ExM workflow to avoid cross talk between NHS-biotin-MA and mCLING. (A) Alternative workflow of land-ExM. (B) i and ii: land-ExM images of U2OS cells stained first with NHS-biotin-MA and then mCLING. iii to v: Magnified images of boxes in i and ii. vi: Normalized intensity profile along the orange line in v. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4.0 (i and ii). 0.5 µm in pre-expansion unit. Linear expansion factor: 4.0 (iii to v). (C) i and ii: land-ExM images of U2OS cells stained first with mCLING and then NHS-biotin-MA. iii to v: Magnified images of orange boxes in i and ii. vi: Normalized intensity profile along the orange line in v. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4 (i and ii). 0.5 µm in pre-expansion unit. Linear expansion factor: 4.0 (iii to v). All images were taken with an Airyscan microscope.