Immunohistochemical Staining of Tumor Samples

For the evaluation of MCDPT, MCAPT, MVD, and EA, a three-layer biotin-avidin-peroxidase system was utilized [17 (link)]. Briefly, 4 μm thick serial sections of formalin-fixed and paraffin-embedded surgically removed tumour samples were deparaffinised. Then, for antigen retrieval, sections were microwaved at 500 W for 10 min, after which endogenous peroxidase activity was blocked with 3% hydrogen peroxide solution. Next, adjacent slides were incubated with the monoclonal antibodies anti-CD31 (clone JC70a; Dako) diluted 1 : 40 for 30 min at room temperature and anti-tryptase (clone AA1; Dako, Glostrup, Denmark) diluted 1 : 100 for 1 h at room temperature. The bound antibody was visualised using biotinylated secondary antibody, avidin-biotin peroxidase complex, and fast red. Nuclear counterstaining was performed with Gill's haematoxylin number 2 (Polysciences, Warrington, PA, USA). Primary antibody was omitted in negative controls.