Embryonic Tissue Characterization Protocols

Tissue collection, 4% paraformaldehyde fixation, processing, paraffin embedding, whole mount staining for β-galactosidase, and H&E staining were performed using established protocols (Lindsley et al., 2007 (link); Snider et al., 2008 (link), 2009 (link)). Collected tissues were sectioned at 6μm thickness. Immunohistochemistry was performed using ABC kit (Vectorstain) with DAB and hydrogen peroxide as chromogens, as described (Olaopa et al., 2011 (link)). The following primary antibodies were used: phospho-SMAD1/5/8 (1:40000, Cell Signaling), α-Smooth muscle actin (1:5000, Sigma), MF20 (1:100, Developmental Studies Hybridoma Bank) and PECAM-1 (1:200, BD Biosciences Pharmingen). Both MF20 and phospho-SMAD1/5/8 antibodies required 10min antigen retrieval (DAKO). Antibody diluent (DAKO), without primary antibody, was used for negative controls. For each assay, whole embryos and/or serial sections were examined for at least three individual embryos of each genotype at each stage of development. Wildtype littermates and Nkx2.5^Cre only embryos were always used as age-matched control samples.