Immunohistochemical Analysis of Liver Tissue

Sections from the left lateral lobe and caudate of the liver were subjected to immunohistochemistry (IHC) with primary antibodies as listed in Supplemental Table S2. After dewaxing and antigen retrieval, tissue sections were immunostained in a Histostainer system (Nichirei Biosciences, Tokyo, Japan) as described previously^{71 (link)}. Briefly, sections were treated with 5% skimmed milk in phosphate buffered saline (PBS) for 10 min, with each primary antibody at room temperature for 1 h, with 3% H₂O₂ in PBS for 15 min, and with horseradish peroxidase-conjugated secondary antibody (Histofine Simple Stain MAX PO; Nichirei Biosciences) at room temperature for 30 min. Positive reactions were visualized with 3,3′-diaminobenzidine (DAB substrate kit; Nichirei Biosciences). After immunohistochemistry, sections were stained with Perls solution for detection of tissue iron.