For the evaluation of MCDPT, PAR-2-MVD, and C-MVD, a three-layer biotin–avidin–peroxidase system was utilized.37 (link) Briefly, 4 μm thick serial sections of formalin-fixed and paraffin-embedded tumor samples and adjacent normal liver tissue were cut. Sections were then microwaved at 500 W for 10 minutes, after which endogenous peroxidase activity was blocked with 3% hydrogen peroxide solution. Tumor sections were incubated with the following primary antibodies: antitryptase (clone AA1; Dako, Glostrup, Denmark) diluted 1:100 for 1 hour at room temperature, anti-PAR-2 (C-17, sc-8205; Santa Cruz Biotechnology, Dallas, TX, USA) diluted 1:50 for 1 hour at room temperature, and anti-CD34 antibody (QB-END 10; Bio-Optica, Milan, Italy) diluted 1:50 for 1 hour at room temperature as a pan-endothelial marker, respectively. The bound antibody was visualized using a biotinylated secondary antibody, an avidin–biotin peroxidase complex and liquid permanent red (LPS, K0640; Dako). Nuclear counterstaining was performed with Gill’s hematoxylin no 2 (Polysciences, Warrington, PA, USA). The primary antibody was omitted in negative controls.