Histological Analysis of Tissue Samples

The harvested cell aggregate, native and decellularized tissue sections from porcine heart and TA muscle from rat were fixed in 4% phosphate-buffered paraformaldehyde for 24 hours, embedded in paraffin. Eight-micrometer-thick serial sections were cut from the paraffin-embedded blocks and underwent H&E staining and Masson’s Trichrome staining. Immunohistochemical staining was performed using standard procedures as described previously [35 (link), 36 (link)]. Briefly, sections were incubated with primary antibodies as follows: anti-Col-I (1:200; Santa Cruz, USA), anti-fibronectin (1:200; Abcam) and anti-integrin-β1 (1:200; Abcam). The same source IgG was used for the negative control instead of the primary antibodies. Biotinylated secondary antibodies (1:1000) were purchased from Sigma-Aldrich. The stained sections were observed using the light microscope (Nikon, Japan). The photographs were evaluated by Image-Pro Plus 6.0 (Media Cybernetics, USA) from three randomly selected views of each specimen.