Free-floating sections were selected based on previous FISH labeling to provide verification of common protein marker identification. Sections selected for KChIP1 were rinsed in Phosphate Tris (PT, pH 7.2-7.4) buffer and Phosphate-Buffered Saline with 0.2% Triton (PBST, pH 7.2-7.4) respectively. They were then moved into a 0.5% H2O2 buffer for 10 minutes followed by a series of rinses before incubating in a 10% Normal Horse Serum (NHS, Vector, S-2000) solution for one hour. The tissue was transferred to primary antibody solution (NeuroMab, Anti-KChIP1, clone K55/7, 1:200) for overnight incubation at 4°C. The next day, sections were again serially rinsed in respective buffers and incubated in secondary antibody (Vector, Vectastain ABC, Peroxidase Kit, PK-4002, 1:200) for 30 minutes. Sections were again serially rinsed and placed into ABC (Vector, Vectastain ABC, Peroxidase Kit, PK-4002) buffer for one hour incubation before being rinsed. Tissue was placed in DAB substrate solution (3,3’-diaminobenzidine, Vector, SK-4100) until reacted. Following a final serial rinse, sections were mounted for imaging and analysis.
