Fig. 5. Immunolabeling of Kv1.1 subunit in whole-mount cochlear preparations. (A, B) Low-magnification images of cochlear preparations (P9) double-labeled with anti-Kv1.1 (red) and anti-parvalbumin (green). Scale bar: 20 μm. (C, D) High-magnification images of cochlear preparations (P9) double-labeled with anti-Kv1.1 (red) and anti-parvalbumin (green). Scale bar: 5 μm. Yellow arrow: Kv1.1 and parvalbumin-positive terminals. (E~J) Confocal micrographs of cochlear preparations (P15) triple-labeled with anti-Kv1.1 (red), anti-NKA (green) and anti-calretinin (blue). Kv1.1 immunoreactivity is present in the majority of NKA and calretinin-positive dendritic terminals contacting onto inner hair cells. Yellow arrow: Kv1.1, NKA and calretinin-positive terminals. White arrow: Kv1.1 and NKA-positive but calretinin-negative terminals. Yellow arrowhead: NKA-positive but Kv1.1 negative terminals. Scale bar: 5 μm. (K~M) Maximum projection images from a confocal z stack show abundant Kv1.1 immunoreactivity in most NKA-positive dendritic segments. (N) Co-localized voxels exhibiting all co-localization result between Kv1.1 and NKA voxels. Scale bar: 5 μm. (O) 2D scatter plot of Kv1.1 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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