Immunohistological Analysis of Fabricated Cell Sheets

Immunohistology was performed according to methods described in our previous article^{30 (link)}. Fabricated cell sheets were fixed in 4% paraformaldehyde (Wako Pure Chemical Industries), dehydrated, embedded in paraffin, sectioned (4 μm) and mounted on glass slides. The slides were de-paraffinized, and antigen retrieval was performed by autoclaving with citrate buffer (Histo-VT One, Nacalai Tesque). Peroxidase blocking (Dako) and protein blocking (Nacalai Tesque) were carried out to prevent non-specific reactions. The slides were then incubated overnight at 4 °C in PBS containing primary antibody (Table S2). After washing with PBS, the slides were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (EnVision Detection System, Peroxidase/DAB, Rabbit/Mouse, HRP; K5007; Dako) at room temperature for 1 h. The sections were washed again and treated with 3,3′-diaminobenzidine (K5007; Dako) to visualize the HRP. Nuclei were stained by hematoxylin. De-paraffinized sections were also stained with hematoxylin and eosin (HE) for histological evaluation.