Application of 4 × and 12 × 3D-ExM in visualiz ing kinetochores. (A) The serial z-stack images of kinetochores labeled with CENP-C at metaphase in RPE1 cells were acquired before expansion (pre-3D-ExM) and after expansion (4× and 12× 3D-ExM) with a step size of 300 nm. The blue bounding box indicates a pair of sister kinetochores that assemble on the same chromosome. (B) The serial z-stack images of mitotic RPE1 cells stained with anti-Plk1 antibodies and DNA dye (DRAQ5) were acquired before expansion (pre-ExM) and after expansion (4× and 12× 3D-ExM) with a step size of 400 nm. Arrows indicate a pair of sister kinetochores that assemble on the same chromosome. 60× water objective (NA = 1.20) was used for imaging.