Mz apo stereomicroscope

Embryos were fixed in 4% paraformaldehyde overnight at 4°C. 8–10 μm cryosections or 5-μm (paraffin-embedded) sections were permeabilised in 0.1% Triton X-100 in PBS (PBT). Antigen retrieval was performed in 0.1 M citrate buffer under pressure boiling for 12 min. After blocking, sections were incubated overnight in a primary antibody (5% BSA in PBT), washed with PBS and incubated with a fluorescent secondary for 1 h. Nuclei were visualised by DAPI (4,6-diamidino-2-phenylindol, 0.1 μg ml⁻¹, Roche). Primary antibodies: Meis2 (Novusbio H00004212-M01) 1:2000, Shh (Santa Cruz Biotechnology sc-9024) 1:2000, Goosecoid (Gsc) (R&D Systems AF4086) 1:1000, FoxF1 (R&D Systems AF4798) 1:1000, FoxF2 (R&D Systems AF6988) 1:1000, Pax3 (DSHB), Casp3 (Cell Signalling 9664) 1:1000, phospho-histone 3 (PH3) (Upstate 6-570) 1:2000, phosphoSmad1/5 (Thermo Fisher Scientific) 1:1000. Secondary antibodies: anti-mouse (-rat, -rabbit) Alexa Fluor488 or 594 (Life Technologies). Biotinylated-anti-mouse, -anti-rabbit, -anti-rat (Vector Laboratories), Vectastain ABC Elite kit and ImmPACT DAB substrate (all Vector Laboratories). Images were acquired in Leica MZ APO stereomicroscope with DC200 camera or Olympus SZX9 with DP72 camera. Fluorescence images were acquired in Zeiss AxioZoom V16 and Zeiss AxioObserver Z1 microscopes. Bright-field light images were acquired in Leica DMLB using Zeiss ZEN Blue software.