Immunostaining for Cardiac Markers

As described previously (Khan et al., 2015), immunofluorescence staining was performed to analyze the expression of cardiac markers in aligned coaxial cardiac patches. Briefly, aligned coaxial cardiac patches, after 2 weeks in culture, were washed twice with PBS and fixed with 4% PFA for 10 min at room temperature. The patches were then washed twice with PBS and incubated in blocking buffer (PBS, 5% normal goat serum, and 0.3% Triton X) for 1 h to block non-specific antibody binding. Following this, the patches were incubated with anti-α-sarcomeric actinin (A7811, MilliporeSigma, Milwaukee, WI, United States), anti-GATA4 (PA1-102, Thermo Fisher Scientific, Waltham, MA, United States), anti-Troponin-T (HPA017888, MilliporeSigma, Milwaukee, WI, United States), and anti-Connexin-43 (MAB 3067, MilliporeSigma, Milwaukee, WI, United States) antibodies, overnight at 4°C and after which the patches were washed thrice in PBS, 5 min. each. Cells were then incubated with the corresponding secondary antibodies conjugated either with Texas Red or FITC against rabbit (1:5000, 8889S, Cell Signaling Technology, Danvers, MA, United States) or mouse (1:5000, 4408S, Cell Signaling Technology, Danvers, MA, United States) for 1 h at room temperature in the dark and washed thrice in PBS. Nuclei were counterstained with NucBlue ({"type":"entrez-nucleotide","attrs":{"text":"R37605","term_id":"795061","term_text":"R37605"}}R37605, Invitrogen, Carlsbad, CA, United States). Finally, the patches were washed thrice with PBS, transferred onto slides and mounted with ProLong^TM Glass Antifade mounting medium (Cat# {"type":"entrez-protein","attrs":{"text":"P36984","term_id":"19858950","term_text":"P36984"}}P36984, Invitrogen, Carlsbad, CA, United States). Imaging was performed on a confocal microscope (Olympus FV 1000 spectral, Olympus Corporation, Center Valley, PA, United States) and images were processed using the Olympus FLUOVIEW Ver. 4.2a Viewer.