Apc 102

Mice (8 weeks old) were anaesthetized and transcardially perfused with 4% PFA and 0.1% glutaraldehyde. After post-fixation overnight, vibratome sections (50 μm) containing the cerebellum were washed and then then immunostained for Kv3.3 for 48h at 4°C using an anti-Kv3.3 antibody raised in rabbit (Alomone labs, APC-102). The sections were then incubated with biotinylated goat anti-rabbit secondary antibodies (Vector, 1:250) for 1.5h at room temperature followed by incubation in Avidin biotin-peroxidase (Vector, 1:200) for 1.5h at room temperature. Immunoreactivity was then visualized with a diaminobenzidine (DAB)/glucose oxidase reaction (Shu et al., 1988 (link)). The sections were then osmicated (15 min in 1% osmium tetroxide) and dehydrated in increasing ethanol concentrations. During the dehydration, 1% uranyl acetate was added to the 70% ethanol to enhance ultrastructural membrane contrast. Flat embedding in Durcupan followed dehydration. Ultrathin sections were cut on a Leica ultramicrotome, collected on Formvar-coated single-slot grids, and analyzed with a Tecnai 12 Biotwin electron microscope (FEI).