B m blocking reagent

Mouse embryos were obtained after euthanasia by cervical dislocation (E14.5 of Mkx^+/− mice). The samples were fixed overnight in 4% PFA/PBS and processed for frozen sectioning (16 μm). Sections were treated with 10 μg ml⁻¹ proteinase K (Roche) for 10 min at room temperature (RT) and then acetylated with acetylation solution for 20 min at RT. Pre-hybridization (in × 5 SSC (pH 4.5), 50% formamide, 1% SDS, 50 μg ml⁻¹ yeast tRNA (Roche) and 50 μg ml⁻¹ heparin (Nacalai Tesque)) was performed at 68 °C for 2 h; then, a DIG–RNA probe (500 ng ml⁻¹) was added and hybridized for 14 h at 68 °C. Subsequently, sections were subjected to a series of post-hybridization washes in wash buffers containing formamide, SSC, SDS and 0.05% CHAPS. After blocking with 2% BM Blocking reagent (Roche) containing 0.1% Tween 20 (TBST) for 30 min at RT, embryos were incubated with anti-DIG-AP Fab antibody fragments (Roche) and 1% sheep serum in TBST for 3 h at RT. After a series of washes with TBST, embryos were equilibrated with NTMT (5 M NaCl, 1 M Tris–HCl (pH 9.5), 1 M MgCl₂ and 0.1% Tween 20). Colour development reactions were performed at 4 °C or RT with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Roche).