For in situ hybridization analysis, brains were fixed with 4% paraformaldehyde and 20 μm thick brain sections were prepared using cryo-stat. Antisense riboprobe for full-length KIAA1212 was generated by in vitro transcription and labeled by digoxygenin. Hybridization of the riboprobes on sections was performed at 65°C overnight and followed by washing once with 5X SSC and 1% SDS, then twice with 2X SSC for 30 min each at 65°C. The incubation of alkaline phosphatase-conjugated anti-digoxygenin antibody was performed at 4°C for overnight, and sections were visualized using nitroblue tetrazolium (NBT, 35 μg/ml)/5-bromo-4-chloro-3-indolyl phosphate 2 (BCIP, 18 μg/ml) color reaction at room temperature.
