5 bromo 4 chloro 3 indolyl phosphate 4 nitroblue tetrazolium bcip nbt substrate solution

In situ hybridization was performed as previously described with some modifications^{48 (link)}. Fragments of the coding regions for Sik1 and Sik2 0.7–0.8 kb in length were generated by PCR using mouse brain and BAT cDNA as templates, respectively. The primers used were Sik1-Fw (5′-ATAGA CTGTG ATCTC CACAG CTCAC TT-3′), Sik1-Rv (5′-ACAGG GAGCA AGCAC ATAGG-3′), Sik2-Fw (5′-AACCC CTCCC TTGAG AGTGT-3′) and Sik2-Rv (5′-GGAAG AGTCG CTTCT GTTGG-3′). Sik1 and Sik2 cDNAs were inserted into pGEM-T easy (Promega) and used for digoxigenin (DIG)-labeled probe synthesis. Mice were perfused with PBS followed by 4% paraformaldehyde (PFA), and harvested brains were postfixed in 4% PFA overnight. Forty μm-thick brain sections were treated with 0.3% Triton X-100, digested with 1 μg/ml proteinase K, treated with 0.75% glycine, and then treated with 0.25% acetic anhydride in 0.1 M triethanolamine. After overnight incubation with a DIG-labeled probe at 60 °C, the sections were washed and then incubated with alkaline phosphatase-conjugated anti-DIG Fab fragments (Roche). The reactions were visualized with a 5-bromo-4-chloro-3-indolyl-phosphate/4-nitroblue tetrazolium (BCIP/NBT) substrate solution (Roche). Sik2-deficient mouse brains were used as negative controls^{39 (link)}.