HMGB1 Immunohistochemical staining
Four micrometers of tissue sections were de-paraffinized, rehydrated, and treated with an antigen retrieval solution (10 mmol/L sodium citrate buffer, pH 6.0). The sections were incubated with a dilution of 1:500 rabbit anti-HMGB1 antibody
(Abcam, UK) overnight at 4°C, and then incubated with 1:1000 dilution of biotinylated secondary antibody, followed by avidin-biotin peroxidase complex
(DAKO, Carpinteria, CA) according to the manufacturer's instructions. Finally, tissue sections were incubated with 3', 3'-diaminobenzidine
(Sigma-Aldrich, Co., Ltd) until a brown color developed, and then the sections were counterstained with Harris' modified hematoxylin. In negative controls, primary antibodies were omitted. Hepatocytes that have brown staining in nucleus area represent normal location of HMGB1, while hepatocytes with brown staining in both cytoplasmic and nucleus area were defined as cytoplasmic translocation of HMGB1. At least 10 high-power fields were chosen randomly, and >1000 cells were counted for each section. Values represent percentages of hepatocytes with HMGB1 cytoplasmic translocation in all hepatocytes counted in each group.
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