4.4. 3,3′-Diaminobenzidine (DAB) Staining Immunohistochemistry

Liver paraffin tissue slides (7 µm) were deparaffinized and rehydrated. The deparaffinized slides were treated with 0.3% H₂O₂ (Sigma-Aldrich, St. Louis, MO, USA) for H₂O₂ block and then washed in phosphate-buffered saline (PBS). The slides were incubated in animal serum to block a nonspecific background, applied with primary antibodies (as listed in Table S2), and then rinsed three times with PBS. The probed slides were treated with biotinylated secondary antibodies from the ABC kit (Vector Laboratories, Burlingame, CA, USA), incubated for 1 h in blocking solution, and washed three times using PBS. The slides were developed with DAB substrate for 5 to 15 min, incubated with hematoxylin to cover the section, and then mounted with a coverslip using DPX mounting solution (Sigma-Aldrich, St. Louis, MO, USA). The images were visualized by light microscopy (Olympus Optical Co., Tokyo, Japan), and quantification of the intensity of the brown color was performed using ImageJ software 1.53j (National Institutes of Health, Bethesda, MD, USA).