Fig. 6. Immunolabeling of Kv1.2 subunit in whole-mount cochlear preparations. (A, B) Low-magnification images of cochlear preparations (P9) double-labeled with anti-Kv1.2 (red) and anti-parvalbumin (green). Scale bar: 20 μm. (C, D) High-magnification images of cochlear preparations (P9 rat) double-labeled with anti-Kv1.2 (red) and anti-parvalbumin (green). Scale bar: 5 μm. yellow arrow: Kv1.2 and parvalbumin-positive terminals. (E~J) Confocal micrographs of cochlear preparations triple-labeled with anti-Kv1.2 (red), anti-NKA (green) and anti-calretinin (blue). Kv1.2 immunoreactivity is present in the majority of NKA and calretinin-positive dendritic terminals contacting onto inner hair cells. Yellow arrow: Kv1.2, NKA and calretinin-positive terminals. White arrowhead: fiber only positive for Kv1.2. yellow arrowhead: NKA-positive but Kv1.2- negative terminals. Scale bar: 5 μm. (K~M) Maximum projection images from a confocal z stack show abundant Kv1.2 immunoreactivity in most NKA-positive dendritic segments as well as the regions further along the nerve fibers. (N) Reconstructed image of voxels exhibiting both Kv1.2 and NKA signals. Scale bar: 5 μm. (O) 2D scatter plot of Kv1.2 and NKA-immunoreactivities. Scatter plot information on all the voxels was extracted from the same confocal 3D dataset depicted in K~M. Yellow vertical and horizontal lines indicate the background signals determined by automated threshold function in Imaris software.
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