4.18. Hematoxylin–Eosin (H&E) Staining and Immunohistochemistry

The tissues were fixed in 10% formalin solution, embedded in paraffin, and sectioned to 4 μm. The sections were then stained with H&E. For immunohistochemistry, antigen retrieval was carried out in 10 mM sodium citrate-hydrochloric acid buffer solution. Thereafter, endogenous peroxidase was blocked via a 15-min incubation in 3% H₂O₂ (methanol). Anti p-STAT3, anti-β-TrCP, and anti-β-catenin (1:50 dilution) were used for the immunohistochemical analysis. Tissues were incubated with the primary antibody at 4 °C overnight. After washing with PBS, the slices were incubated with the appropriate secondary antibody for 30 min at 37 °C. Peroxidase activity was revealed using 3,3-diaminobenzidine and counter-stained with hematoxylin. Images were captured using a BDS 200 microscope (CNOPTEC, Chongqing, China) and a camera (Canon, Tokyo, Japan).