Sarcomeric α actin

Tissues were processed as described previously (12 (link)). Briefly, rat hearts were excised after hemodynamic data collection under anesthesia and perfused with 4% paraformaldehyde. Human tissue was fixed with 4% paraformaldehyde. Tissues were cryo-preserved using 30% sucrose and embedded in OCT (TissueTek). Sections were cut to 7µm, using a commercial cryostat, and stained for isolectin B4 (Invitrogen; Carlsbad, CA), α-SMA (Sigma), sarcomeric-α-actin (Santa Cruz Biotechnology; Santa Cruz, CA), anti-human nuclei (Millipore; Billerica, MA), or c-kit⁺ (BD Biosciences) primary antibodies. For Isl-1 staining, the cells were fixed with 4% PFA, permeabilized, overnight incubated with Isl-1 antibody (R & D Systems), incubated with FITC labeled secondary antibody. Cells and tissue sections were counterstained with DAPI (4’,6-diamidino-2-phenylindole) nuclear stain (Sigma; St Louis, MO).

Heart biopsy specimens were fixed in 4% para-formaldehyde and then cryopreserved using 30% sucrose and embedded in OCT (Sakura Finetek, Zoeterwoude, The Netherlands). Sections were cut to 7 μm, using a commercial cryostat, and stained for sarcomeric-α-actin (Santa Cruz Biotechnology, Santa Cruz, CA), transcription factor GATA-4 (Santa Cruz Biotechnology), or c-kit⁺ (BD Biosciences, Franklin Lakes, NJ) primary antibodies. For islet-1 (ISL-1) staining, tissue sections were permeabilized, incubated overnight with ISL-1 antibody (R&D Systems, Minneapolis, MN), and incubated with fluorescein isothiocyanate–labeled secondary antibody. Tissue sections were counterstained with nuclear 4,^′6-diamidino-2-phenylindole stain (Sigma).