Hm 500

After immobilization by cold anesthesia, the brains of embryos were dissected in cold phosphate-buffered saline (PBS) and immersed in 4% paraformaldehyde (PFA) in 0.12 M phosphate buffer (PB) overnight at 4 °C. Adult (P21-30) mice were deeply anesthetized with sodium pentobarbital (Kyoritsu Pharmacy; 100 mg/kg body weight) and perfused transcardially with PBS followed by 4% PFA in 0.12 M PB and immersed overnight in the same fixative at 4 °C. The brains were cryoprotected by immersion in 30% sucrose in 0.1 M PB overnight, embedded in OCT compound (Sakura Finetek) and quickly frozen in liquid-nitrogen cooled isopentane. For embryos, transverse sections were cut on a cryostat (HM500, Zeiss) at 20 or 30 μm and the sections that included ganglionic eminences (GEs) were mounted on slides (Superfrost Plus, Fisher Scientific). For adult mice, serial sections were cut coronally at 50 μm and 80–100 sections were cut rostrocaudally starting from the caudal end of the rhinal incisure. In some animals, sections were cut from the level of the olfactory bulb. Every ten sections were then collected as free-floating sections, subjected to immunostaining and mounted on slides.