We followed a previously established protocol for optimized detection of gene expression through non-radioactive in situ hybridization
[33 (link)]. Briefly, for each brain-derived cDNA clone, we generated digoxygenin-labelled sense and antisense riboprobes, performed in situ hybridization using high-stringency hybridization and wash conditions, and detected cellular labelling by immunohistochemical detection with alkaline phosphatase precipitation. Under these hybridization conditions, sense-strand hybridizations and hybridizations that are performed without probe give no signal. Replicates were run for each probe on at least two adjacent sections (n = 3 brains) for all of the major song nuclei. Resulting high-quality sections were then imaged for digital analysis at 0.42 μm/pixel with an Olympus Nanozoomer. For each nucleus we assessed relative level of brain expression based on visual assessment and a scoring scale from low to high. Genes that we found to be expressed in song nuclei were qualitatively analysed for enrichment (or impoverishment) relative to surrounds (e.g. RA vs. arcopallial shelf) by at least two independent observers.
[33 (link)]. Briefly, for each brain-derived cDNA clone, we generated digoxygenin-labelled sense and antisense riboprobes, performed in situ hybridization using high-stringency hybridization and wash conditions, and detected cellular labelling by immunohistochemical detection with alkaline phosphatase precipitation. Under these hybridization conditions, sense-strand hybridizations and hybridizations that are performed without probe give no signal. Replicates were run for each probe on at least two adjacent sections (n = 3 brains) for all of the major song nuclei. Resulting high-quality sections were then imaged for digital analysis at 0.42 μm/pixel with an Olympus Nanozoomer. For each nucleus we assessed relative level of brain expression based on visual assessment and a scoring scale from low to high. Genes that we found to be expressed in song nuclei were qualitatively analysed for enrichment (or impoverishment) relative to surrounds (e.g. RA vs. arcopallial shelf) by at least two independent observers.
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