For histological analysis of esophageal regeneration, esophageal tissues were excised along with tracheal tissue to support the esophagus. For routine staining, tissues were fixed with cooled acetone. To obtain cryosections, the tissue was perfused with sucrose solution, embedded in optimal cutting temperature compound, and frozen at −80 °C. Tissue sections were then stained with hematoxylin and eosin (H&E) and Masson's trichrome stain. Stained sections were scanned using an Axio Scan.Z1 (Carl Zeiss Microscopy).
For immunohistochemical analysis, the tissue samples were fixed in 4% PFAPFA overnight and embedded in paraffin. To detect smooth muscle regeneration and vascularization, the esophageal tissue sections were stained with anti-SM22α, anti-IL-B4, anti-vimentin, and anti-desmin antibodies. Epithelium of esophageal tissues were stained with anti-cytokeratin 14, and Macrophages were stained with anti-CD68 antibody. The secondary antibodies used were goat anti-mouse antibody conjugated with Alexa Fluor 488 or 568 and streptavidin conjugated to Alexa Fluor 488, and DAPI was used to stain the nuclei. The stained sections were visualized under a laser confocal microscope (Olympus FluoView FV1000) and an Axio Scan.Z1 (Carl Zeiss Microscopy). The fluorescence levels were quantified in a high-power field using ImageJ software.
For immunohistochemical analysis, the tissue samples were fixed in 4% PFAPFA overnight and embedded in paraffin. To detect smooth muscle regeneration and vascularization, the esophageal tissue sections were stained with anti-SM22α, anti-IL-B4, anti-vimentin, and anti-desmin antibodies. Epithelium of esophageal tissues were stained with anti-cytokeratin 14, and Macrophages were stained with anti-CD68 antibody. The secondary antibodies used were goat anti-mouse antibody conjugated with Alexa Fluor 488 or 568 and streptavidin conjugated to Alexa Fluor 488, and DAPI was used to stain the nuclei. The stained sections were visualized under a laser confocal microscope (Olympus FluoView FV1000) and an Axio Scan.Z1 (Carl Zeiss Microscopy). The fluorescence levels were quantified in a high-power field using ImageJ software.