Rabbit anti pax2

Mouse embryos were dissected to extricate the brains, which were fixed in 4% PFA at +4°C for 18-36h, and kept in 30% sucrose at 4°C until saturated, upon which they were frozen in OCT Tissue Tek (Sakura Finetek, Alphen aan den Rijn, Netherlands) and stored at -80°C. Cryosections (30 µm) were treated with 4% PFA for 15 min at room temperature, blocked and processed with primary antibodies in PBS with 0.2% Triton-X100 and 4% horse serum overnight at +4°C. Secondary antibodies, conjugated with AMCA, FITC, Rhodamine-RedX, Cy5 (Jackson ImmunoResearch, West Grove, PA, USA), or AFD555, AF568 or AF647 (Thermo Fisher Scientific, Waltham, MA, USA), were used at 1:200. Slides were mounted in Vectashield (Vector Laboratories, Burlingame, CA, USA). DAPI was included in the secondary antibody solution. Primary antibodies: Goat anti-Sox2 (1:250, #SC-17320, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Rabbit anti-H3K27me3 (1:500, #9733, Cell Signaling Technology, Leiden, Netherlands). Rat anti-PH3-Ser28 (1:1,000; Cat.no. ab10543). Rabbit anti-Pax2 (1:100, #ab232460), rabbit anti-TFAP2b (1:100, #ab221094), rabbit anti-Orexin A (Hcrt) (1:1,000, ab6214), rabbit anti-Avp (1:500, #ab213708, Abcam, Cambridge, UK).