Nucleotide sequences were determined MK-1775 mw using a Dye Terminator Cycle Sequencing kit (Applied Biosystems, Tokyo, Japan) and the ABI 3730xl DNA sequencer (Applied Biosystems). Sequences were compared with the National Center for Biotechnology Information (NCBI) nucleotide database using the Basic

Local Alignment Search Tool (BLAST) program. In situ hybridization was performed as described by Matsunaga and Okanoya (2008) and Kato and Okanoya (2010). Common marmosets were removed from the family cage, anesthetized with a mixture of ketamine and xylazine, and then killed by exsanguination. The marmoset could freely express calls before anesthesia. Brains were quickly dissected, frozen on dry ice in an embedding medium (Tissue-Tek; Sakura Finetek, Torrance, CA, USA), and then cut into 20-μm-thick coronal sections on a cryostat. Digoxigenin (DIG)-labeled probes were generated using the original cDNA PCR products as templates. PCR products were generated using universal primers (SP6 primer, 5′-TAATACGACTCACTATAGGG-3′; and T7 primer, 5′-TAATACGACTCACTATAGGG-3′) and purified using the Wizard® SV Gel and PCR Clean-Up System (Promega). Probes were synthesized using T7 or SP6 RNA polymerase (Roche Diagnostics, Switzerland) and a DIG-labeling mix (Roche Diagnostics). Torin 1 research buy Glass-mounted brain tissue sections were fixed in 4% paraformaldehyde for 10 min

and then rinsed three times with phosphate-buffered saline (PBS) for 5 min each. Sections were acetylated for 10 min in distilled water containing 1.35% triethanolamine, 0.25% acetic anhydride, and 0.065% HCl, and then incubated in PBS containing 1% Triton X-100 for 30 min. Next, sections were rinsed three times with PBS for 5 min each time, and then incubated

in a hybridization solution of RNase-free water containing 50% formamide, 5 × saline sodium citrate (SSC), 5 × Denhardt’s solution, 0.24 mg/mL yeast tRNA, 0.5 mg/mL salmon sperm DNA, and labeled probes. The sections were coverslipped in a humid box and incubated overnight at 72 °C. The next day, coverslips were removed by placing sections in pre-warmed 5 × SSC at 72 °C, and then the sections washed by sequential incubations for 2 h at 72 °C in 0.2 × SSC, for 5 min at room temperature (RT) in Adenosine triphosphate 0.2 × SSC, and for 5 min at RT in buffer 1 (RNase-free water containing 0.1 M Tris–HCl (pH 7.5), 0.15 M NaCl, and 0.001% Tween 20). Sections were then incubated for 1 h in a blocking solution consisting of buffer 1 supplemented with 10% sheep serum. Next, sections were incubated overnight at RT with an alkaline phosphatase-conjugated anti-DIG antibody (1:5000 dilution in blocking buffer; Roche Diagnostics). The following day, sections were washed three times in buffer 1 for 5 min each at RT, and then incubated for 15 min in buffer 2 (0.1 M Tris–HCl (pH 9.5), 0.1 M NaCl, 50 mM MgCl2, 0.001% Tween 20, and 0.