Histology and immunohistochemical staining of 4-μm-thick tissue sections were performed as described previously [32 ,33 (link)] using the following primary antibodies: mouse monoclonal anti-α-SMA antibody (Sigma-Aldrich, St. Louis, MO, USA); rabbit polyclonal anti-FSP-1 antibody (Abcam, Cambridge, UK); rabbit polyclonal F4/80 antibody (Abcam); rabbit polyclonal anti-type I collagen antibody (Abcam); rabbit polyclonal anti-type III collagen antibody (Chemicon International, Temecula, CA, USA); rabbit polyclonal anti-TGF-β1 antibody (Sigma-Aldrich); rabbit polyclonal anti-GLP-1R antibody (Bioss, Woburn, MA, USA).
Type I and III collagen-stained areas were assessed in predetermined fields (×200 magnification) of the submesothelial compact zone, captured by a digital camera, and analyzed using ImageJ software (version 1.44p; National Institutes of Health, Bethesda, MD, USA). The numbers of cells positive for α-SMA, FSP-1, TGF-β1, F4/80, and GLP-1R in the submesothelial compact zone were counted in nine fields at ×200 magnification.
Type I and III collagen-stained areas were assessed in predetermined fields (×200 magnification) of the submesothelial compact zone, captured by a digital camera, and analyzed using ImageJ software (version 1.44p; National Institutes of Health, Bethesda, MD, USA). The numbers of cells positive for α-SMA, FSP-1, TGF-β1, F4/80, and GLP-1R in the submesothelial compact zone were counted in nine fields at ×200 magnification.