Qb end 10

For the evaluation of MCPT and MVD a three-layer biotin-avidin-peroxidase system was utilized [33] (link). Briefly, 4 µm-thick serial sections of formalin-fixed and paraffin-embedded tumour sample and adjacent normal mucosa were cut. Sections were then microwaved at 500 W for 10 min, after which endogenous peroxidise activity was blocked with 3% hydrogen peroxide solution. Tumour sections were incubated with an anti-tryptase (clone AA1; Dako, Glostrup, Denmark) diluted 1∶100 for 1 h at room temperature and an anti-CD34 antibody (QB-END 10; Bio-Optica Milan, Italy) diluted 1∶50 for 1 h at room temperature as a pan-endothelial marker respectively. Adjacent normal mucosa sections were incubated with an anti-tryptase (clone AA1; Dako, Glostrup, Denmark) and then processed in the above manner. Tumour sections were also incubated with an anti-VEGF (Clone VG-1) (Dako Glostrup, Denmark), diluted to 1∶200 for 1 h at room temperature. The bound antibody was visualized using a biotinylated secondary antibody, avidin-biotin peroxidise complex and fast red. 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 TAMs, TAMIA, MVD, EA and CCP‐VEGF‐A, a three‐layer biotin–avidin–peroxidase system was utilized 49. Briefly, 4‐μm‐thick serial sections of formalin‐fixed and paraffin‐embedded tumour sample were cut. Sections were then microwaved at 500 W for 10 min, after which endogenous peroxidise activity was blocked with 3% hydrogen peroxide solution. Tumour sections were incubated with the primary anti‐CD68 antibody (Clone KP1; Dako, Glostrup, Denmark) diluted 1:100 for 1 hr at room temperature, the primary anti‐CD34 antibody (QB‐END 10; Bio‐Optica Milan, Italy) diluted 1:50 for 1 hr at room temperature as a pan‐endothelial marker, respectively, and, finally with the primary anti‐VEGF‐A antibody (Neomarker, Freemont, CA, USA) diluted 1:50 for 1 hr at room temperature. The bound antibody was visualized using a biotinylated secondary antibody, avidin–biotin peroxidise complex and fast red. Nuclear counterstaining was performed with Gill's haematoxylin no.2 (Polysciences, Warrington, PA, USA). The primary antibody was omitted in negative controls.