Embryo In Situ Hybridization and Immunofluorescence

For in situ hybridization (ISH), embryos were fixed in 4% PFA in PBS overnight at 4°C, dehydrated in methanol and stored at −20°C. ISH was performed using antisense riboprobes as deSCRIBed previously^{38 (link)}. Immuno-fluorescence was carried out as previously deSCRIBed^{38 (link)}. Primary antibodies were used: COLL-IV (1:300, Millipore), FN-1 (1:300, Rockland), FOXA2 (1:1000, Abcam), LAMA-1 (1:300, Sigma), LAMB-1 (1:300, Abcam) and SOX17 (1:1000, R&D Systems) ITGA5 (1:300, Santa Cruz), E-CAD (1:300, Sigma), N-CAD (1:300, Santa Cruz), and SCRIB (1:200, Santa Cruz). Secondary Alexa-Fluor conjugated antibodies (Invitrogen) were used at a dilution of 1:1000. DNA was visualized using Hoechst-33342 (5 μg/mL, Molecular Probes). For cryosections, fixed embryos were taken through a sucrose gradient, embedded in O.C.T. (Tissue-Tek) and sectioned at 12 μm on a cryostat (CM3050S, Leica).