After the animals were sacrificed, the duodenum was resected, and the ESD areas were harvested for the evaluation and measurement. The specimens were fixed in 10% formalin and embedded in serial-section paraffin blocks, after which they were carefully cut from the paraffin-embedded blocks and stained with hematoxylin and eosin (H&E).
To confirm the attachment of the myoblast sheets to the surface of the duodenum at the ESD serosal site, immunostaining examinations were performed. The sections were deparaffinized with xylene and rehydrated in 99% ethanol. After treatment with preheated a pH 6.0 target retrieval solution (Agilent Technologies, Santa Clara, CA, USA), the slides were made to be with the water for 20 min. The sections were rinsed in phosphate-buffered saline and soaked for 10 min at room temperature in peroxidase-blocking solution (Agilent Technologies). Monoclonal mouse anti-desmin antibody (Agilent Technologies) was used as the primary antibody. The sections were incubated at 4°C overnight. After washing, the sections were treated with horseradish peroxidase–labeled polymer conjugated to goat anti-mouse (Agilent Technologies) at room temperature for 30 min. Sections were then stained with 3,3-diaminobenzidine tetrahydrochloride containing hydrogen peroxide (Agilent Technologies).
To confirm the attachment of the myoblast sheets to the surface of the duodenum at the ESD serosal site, immunostaining examinations were performed. The sections were deparaffinized with xylene and rehydrated in 99% ethanol. After treatment with preheated a pH 6.0 target retrieval solution (Agilent Technologies, Santa Clara, CA, USA), the slides were made to be with the water for 20 min. The sections were rinsed in phosphate-buffered saline and soaked for 10 min at room temperature in peroxidase-blocking solution (Agilent Technologies). Monoclonal mouse anti-desmin antibody (Agilent Technologies) was used as the primary antibody. The sections were incubated at 4°C overnight. After washing, the sections were treated with horseradish peroxidase–labeled polymer conjugated to goat anti-mouse (Agilent Technologies) at room temperature for 30 min. Sections were then stained with 3,3-diaminobenzidine tetrahydrochloride containing hydrogen peroxide (Agilent Technologies).