Immunohistochemical Staining of Protein Targets

Immunohistochemical (IHC) staining was performed using the BenchMark ULTRA slide staining system (Ventana Medical Systems, USA). Briefly, TMA tissue sections were deparaffinized and rehydrated with EZprep concentrate (10×) solution (Ventana Medical Systems). Antigen retrieval was performed with cell conditioning 1 (Ventana Medical Systems) at 95°C for 30 min. Slides were then treated with iView DAB (3,3'-diaminobenzidine tetrahydrochloride) Inhibitor (Ventana Medical Systems) and incubated with 100 µL primary antibody (1∶100) for 90 min. The following primary antibodies were used for IHC staining: pAMPK (Thr 172), pACC (Ser 79), and phosphorylated extracellular signal-regulated kinase (pErk) (Thr 202/Tyr 204) (All Cell Signaling Technology; Danvers, MA, USA) and immunoglobulin G (clone SP3) (Thermo Fisher Scientific, USA). These antibodies were chosen because they have been investigated in other studies [15] (link)–[17] (link). Bound antibody was detected using the iView DAB Detection Kit (Ventana Medical Systems) according to the manufacturer’s instructions.