Anti rabbit horseradish peroxidase labeled antibody

The tumor tissue was fixed, embedded in paraffin, and cut into 5‐μm‐thick sections, which were placed onto glass slides. The sections were then deparaffinized, rehydrated, and subjected to antigen retrieval. Next, the slides were blocked with goat serum and incubated with rabbit mAbs (1 : 200 dilution) against CD3 (ab135372; Abcam) or granzyme and then with a horseradish peroxidase‐labeled anti‐rabbit antibody (46890; Cell Signaling). Finally, the slides were developed with diaminobenzidine and counterstained in hematoxylin. Target protein expression was scored semiquantitatively according to a routine IHC grading system, and the values were used for statistical analysis.

Liver tissues were homogenized in RIPA buffer (Thermo Fisher Scientific) containing 5 mM dithiothreitol, 200 mM phenylmethylsulfonyl fluoride, protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA), 5 mM EDTA, 10 mM NaF, and 10 mM Na₃VO₄. After sonication, liver proteins were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electro-transferred to membranes using a Trans-Blot Turbo system (Bio-Rad, Hercules, CA, USA). After blocking with Blocking One (Nacalai Tesque, Kyoto, Japan), the membrane was incubated with rabbit anti-LPL antibody (1:1000; catalog no. GTX101125; GeneTex, Irvine, CA, USA), anti-CPT1 antibody (1:4000; catalog no. 15184-1-AP; Proteintech Group Inc, Rosemont, IL, USA), anti-ACOX1 antibody (1:4000; catalog no. 10957-1-AP; Proteintech), anti-citrate synthase antibody (1:4000; catalog no. 16131-1-AP; Proteintech), or mouse anti-β-actin antibody (1:5000; catalog no. A5441; Sigma-Aldrich; 1:5000; catalog no. 60008-1-Ig; Proteintech), followed by incubation with horseradish peroxidase-labeled anti-rabbit antibody (1:5000; catalog no. #7074) or anti-mouse IgG antibody (1:5000, catalog no. #7076; Cell Signaling Technology, Danvers, MA, USA). The western blot results were analyzed using the NIH image analysis software ImageJ (NIH, Bethesda, MD, USA). The protein levels were normalized to that of β-actin.