Immunohistochemical Analysis of α-SMA and TGF-β1

With the standard protocol being followed prior to HE staining described above, the sections were subjected to antigen retrieval and blocking as previously described [15 (link)]. For the immunohistochemical analysis, the sections were initially incubated with mouse anti-α-SMA antibody (Abcam, Cambridge, MA, USA) or mouse anti-TGF-β1 antibody (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 4°C overnight and were then incubated with HRP-conjugated goat antimouse secondary antibody (Abcam, Cambridge, MA, USA) at 37°C for 30 min. Positive staining was detected with HRP-conjugated streptavidin, visualized with 3,3′-diaminobenzidine, and counterstained with hematoxylin. Finally, the sections were mounted and cover-slipped, and images of five representative fields at ×400 magnification were captured by the Leica QWin Plus v3 software (Leica Microsystems, Cambridge, UK). The sizes of the study areas and the integrated optical density (IOD) of the positive stains were measured using Image-Pro Plus 5.1 (Media Cybernetics, Rockville, MD, USA). The expression levels of the proteins were expressed as the mean optical density (MOD; MOD = IOD per unit of study area).