E14.5 embryonic hearts were dissected and fixed for 30 min in 4% paraformaldehyde followed by a 1 hr wash in distilled water and secondary fixation overnight. Fixed samples were dehydrated and embedded in modified JB4 methacrylate resin (Weninger et al., 2006 (link)) and sectioned at 2 μm. HREM imaging (isometric resolution of 2 μm) used a Hamamatsu Orca-HR camera. Data sets were normalized and subsampled prior to 3D volume rendering using Osirix v5.6 (Rosset et al., 2004 (link)). As the expected CHD were not fully penetrant, a minimum of 17 E14.5 mutant embryos were compared to littermate controls. Phenotype analysis was performed blind for genotype, and classification of type of CHD was carried out as previously described (Dunlevy et al., 2010 (link)). For dual-wavelength HREM, a conventional Xgal reaction was performed followed by 4% paraformaldehyde fixation, dehydration and embedding, and imaging was carried out using a Jenoptik ProgRes C14 camera with dual filter (59022bs, Chroma Technology Corp). Image analysis of the DMP was done using ITKsnap 2.4.0 and Volocity 6.2.1 software packages. Volocity was used to calculate the volume of the DMP and the shape factor (shape factor is 1 for a perfect sphere and <1 for more irregular shapes). A minimum of 9 biological replicates per group was analyzed and analysis of the DMP was performed blind for genotype.
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