Nanozoomer

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.

Timed matings were set up to collect the embryos varying from embryonic day E8.5 to E13.5. Embryo genotypes were determined by PCR from yolk sac DNA. Embryos were fixed in 4% paraformaldehyde and stained overnight in 5-bromo-4-chloro-3-indolyl--D-galactoside-containing solution to visualize β-galactosidase activity derived from the lacZ gene tag in the Tarbp2-targeted mutation. For tissue sections, embryos were frozen in optimum cutting temperature (OCT) compound (Sakura Finetek USA Inc.) and cryostat sectioned. Sections were fixed (0.2% gluteraldehyde) and stained as above. Sections were postfixed, counterstained with Nuclear Fast Red, mounted, and imaged under an Olympus Nanozoomer with the desired magnification.

Sections were examined with a bright field virtual slide system (Hamamatsu Photonics, NanoZoomer), a fluorescent microscope (Olympus, BX51WI), and a confocal laser microscope (Olympus, FV1200).