Colocalization between Cav1.3 channels and ribbons. A, B, 3D stack reconstruction of confocal images of utricle (A, striolar region) and OC (apical region, B). Organs (P14) were labeled for F-actin (pink), Cav1.3 (green), and CtBP2 (red ribbons). Afferent fibers were labeled with Anti-NF200 (blue). Asterisks indicate VHC-I surrounded by large calyceal nerve terminals. Scale bar, 6 μm. An example of a Cav1.3 channel cluster (green) and its ribbon (red) is shown for VHC-I (A′) and IHCs (B′). Scale bar, 0.25 μm. C, D, Colocalization between Cav1.3 and ribbon (CtBP2-ribeye) was assessed by plotting the fluorescence intensity profile from a line scan (A′, B′, white dashed line). An example of fluorescence intensity profile is shown for VHC-I (C) and IHCs (D). E, Colocalization analysis using JACoP plugin in ImageJ. Gaussian fit of the occurrences of center mass distance measured between ribbon and Cav1.3 VHC-I (167.77 ± 4.53 nm, n = 83) and IHCs (308.34 ± 4.68 nm, n = 106) suggested a tighter organization of Cav1.3 channels and ribbons in VHC-I.