3 3 diaminobenzidine solution

Deparaffinization sections were washed thrice for 5 min each with phosphate buffered saline (PBS) (pH 7.4). For antigen activation, Proteinase K (Worthington Biochemical Co., Lakewood, NJ, USA)/distilled water (0.2 mg/mL) was added dropwise to the sections, which were then incubated for 15 min. After rewashing with PBS, endogenous peroxidase activity was blocked by BLOXALL blocking solution (Vector Laboratories, Newark, CA, USA) for 10 min. Blocking used 0.05% normal goat serum/PBS solution for 1 h. After rewashing again with PBS, the following primary alternatives were reacted at 4 °C overnight: antitumor necrosis factor-alpha (TNF-α), rabbit polyclonal antibody (1:200 dilution, AF7014; Affinity Biosciences, Solon, OH, USA), and anti-interleukin-6 (IL-6) rabbit polyclonal antibody (1:100 dilution, ab6672; Abcam, Cambridge, MA, USA). Subsequently, the streptavidin-biotin-peroxidase complex technique was performed using an ABC kit (Vector Laboratories). After rewashing with PBS, the sections were colored brown using a 3,3-diaminobenzidine solution (NICHIREI Biosciences, Tokyo, Japan). Nuclei were counterstained with Mayer’s hematoxylin. Immunohistochemically (IHC) stained images were calculated as the ratio of positive cells per unit area using the Fiji image analysis software [18 (link)].

Hepatic tissue specimens were fixed with Carnoy’s fixative in 10% methanol overnight. Fixed liver tissues were embedded in paraffin and cut to a thickness of 4 µm. The sections were mounted on adhesive glass slides (Matsunami Glass Ind., Osaka, Japan) and deparaffinized with xylene and ethanol. Hepatic histological changes were assessed using HE staining and Sirius Red staining (Polysciences, Inc., Warrington, PA, USA). For IHC staining, endogenous peroxidase activity and non-specific antigens were blocked with 10% H₂O₂ and Serum-Free Ready-to-Use Protein Block (Dako, Santa Clara, CA, USA), respectively, followed by incubation of the sections with antibodies against PKM2, F4/80 (Cell signaling Technology, Inc., Danvers, MA, USA), PKLR, and CD68 (Abcam, Cambridge, MA, USA) overnight at 4 °C. Sections were then washed with PBS and incubated with EnVision Dual Link System-HRP (Dako) as the secondary antibodies at room temperature for 30 min. Immunoreactions were visualized using 3,3-diaminobenzidine solution (Nichirei Biosciences Inc., Tokyo, Japan) and counterstained with hematoxylin. Finally, the sections were dehydrated and mounted. Images were taken with a BZ-x700 microscope (KEYENCE, Tokyo, Japan). The antibodies used are described in Table S1.