Serial coronal sections (12 μm thick) were cut from 31 whole zebra finch brains using a cryostat, mounted on positively charged slides (SuperfrostPlus®, Fisher Scientific, Pittsburgh, PA, United States), dried, and stored at −80°C until use. The brain sections were collected in such a way that consecutive sections were mounted on 18 parallel slides. For in situ hybridisation, [35S]UTP-labelled riboprobes were generated from the DNA probes using a MAXIscript transcription kit (Ambion, Austin, TX, United States).
The preparation of tissue was performed using an mRNAlocator Kit (Ambion), according to the manufacturer’s instructions. Tissue was prepared using an mRNA-locator Kit (Ambion) according to manufacturer’s instructions. For hybridisation, we used 80 μl hybridisation buffer and 1 million DPM of labelled probe per slide. Washing procedures included a 30 min incubation in RNase A, followed by decreasing concentrations of sodium-citrate buffer (pH = 7.4) at room temperature, and then at 65°C. After drying, slides were dipped in NTB nuclear track emulsion (Eastman Kodak, Rochester, NY, United States), stored for 3 weeks at 4°C for autoradiography, developed with Kodak Dektol developer, fixed with Kodak fixer, counterstained with Giemsa, and coverslipped with Cytoseal 60 (Stephens Scientific, Riverdale, NJ, United States).
The preparation of tissue was performed using an mRNAlocator Kit (Ambion), according to the manufacturer’s instructions. Tissue was prepared using an mRNA-locator Kit (Ambion) according to manufacturer’s instructions. For hybridisation, we used 80 μl hybridisation buffer and 1 million DPM of labelled probe per slide. Washing procedures included a 30 min incubation in RNase A, followed by decreasing concentrations of sodium-citrate buffer (pH = 7.4) at room temperature, and then at 65°C. After drying, slides were dipped in NTB nuclear track emulsion (Eastman Kodak, Rochester, NY, United States), stored for 3 weeks at 4°C for autoradiography, developed with Kodak Dektol developer, fixed with Kodak fixer, counterstained with Giemsa, and coverslipped with Cytoseal 60 (Stephens Scientific, Riverdale, NJ, United States).