Lama 1

Following PBS and PFA perfusion, mouse tissues were incubated with 30% sucrose solution overnight, embedded in frozen section media and snap frozen on dry ice. 16 μm sections were cut on a Micron cryostat for histology staining and quantifications. The following antibodies were used for immunostainings: Podocalyxin (AF1556 R&D; 1:250), Collagen IV (2150-1470 Serotec; 1:250), SPP1 (HPA027541, Atlas antibodies 1:250), COL6A1 (HPA019142, Atlas antibodies, 1:250) and LAMA1 (L9393, Sigma, 1:500). Secondary goat antibodies conjugated to Alexa Fluor 488, 555, 594 or 647 were purchased from Life Technologies and used at 1:500. Images were acquired using Zeiss LSM 700 confocal microscope and Zen software.

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).