NM-II immunostaining closely correlates with the periodic actin rings along the axon. (a) DIV14 rat hippocampal neurons were stained for endogenous F-actin (Phalloidin) and NM-IIB and imaged with dual-color 3D SIM. Boxed regions are shown with individual z-stack planes; periodic structure of NM-II and actin are indicated with arrows (green) and spots (red), respectively. (b) The NM-II and actin structures resolved with 3D SIM (right) were rendered into surface (right) using Imaris; boxed regions are magnified to show the accuracy of the rendering. The colocalization of the bracketed region is shown in the bottom panels. (c) Two-color SIM images of endogenous F-actin (phalloidin) and NM-II along the axons of control and Triton X-100–extracted axons. Scale bars = 1 µm. (d–f) Quantification of the percentage of axons bearing periodic actin rings (d), NM-II puncta number (e), and the percentage change in these two parameters (f) in control and Triton X-100–extracted axons (+Triton). Data represent mean ± SEM; n = 10 cells (control) and 9 cells (Triton X-100) from three independent cultures (**, P < 0.01; ***, P < 0.001, two-tailed unpaired t test).