Anti par 2 c 17 sc 8205

For the evaluation of TMCD, PAR-2-MVD and C-MVD, a three-layer biotin–avidin–peroxidase system was utilized [Ranieri et al. 2007 (link)]. Briefly, 6 μm-thick serial sections of formalin-fixed and paraffin-embedded tumour sample were cut. Sections were then microwaved at 500W for 10 minutes, after which, endogenous peroxidise activity was blocked with 3% hydrogen peroxide solution. Tumour sections were incubated with the following primary antibodies: antitryptase (clone AA1; Dako, Glostrup, Denmark) diluted 1:100 for 1 hour at room temperature specific for MC identification; anti-PAR-2 (C-17; sc-8205, Santa Cruz Biotechnology, Texas, 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, avidin–biotin–peroxidase complex and liquid permanent red (LPS, K0640, Dako, Glostrup, Denmark). Nuclear counterstaining was performed with Gill’s haematoxylin no.2 (Polysciences, Warrington, PA, USA). The primary antibody was omitted in negative controls.

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.