Cardiac troponin 1

Slides were de-waxed with Histoclear and rehydrated through a series of ethanol washes. Following washes in PBS, antigen retrieval was performed by boiling slides in citrate buffer (0.01 mol/L) for 10 minutes. Samples were blocked in 10% FCS and then incubated either overnight at 4°C, or at room temperature for 2 hours with the following antibodies: E-cadherin, β-catenin, N-Cadherin (BD Transduction Laboratories), fibronectin, Scrib, PKCζ (Santa Cruz), Isl1, MF20 (Developmental studies Hybridoma Bank, University of Iowa), GFP, alpha smooth muscle actin (Abcam), gamma tubulin, laminin (Sigma), cardiac troponin I (HyTest), desmin (Millipore). For immunofluorescence, samples were incubated at room temperature for two hours, with secondary antibodies conjugated to either Alexa 488 or Alexa 594 (Life Technologies). Fluorescent slides were washed then mounted with Vectashield Mounting medium with DAPI (Vector Labs). For non fluorescent staining, samples were incubated with biotinylated secondary antibodies for 1 hour, then with AB complex (Vector labs) for a further hour. Slides stained with DAB were washed then counter-stained with 5% methyl green. After dehydration in 100% butanol and Histoclear, slides were mounted using Histomount.