Ventana benchmark ultra staining system

The Roche Ventana BenchMark Ultra staining system and the ultraView DAB detection kit were used for all immunostainings. After warming the slides up to 75 °C, deparaffinizing and pretreating them (not for αSMA), the staining kit was applied. Having added 3% H₂O₂, the slides were incubated with the primary antibody. To visualize the bound structures, the secondary antibody, diaminobenzidine (DAB), H₂O₂, and copper were applied. Having counterstained the sections and washed them twice, they were dehydrated through increasing alcohol concentration and covered with Entellan® (Merck Millipore, Darmstadt, Germany).
Tumor specimens were stained for Mib-1/Ki67 (proliferation, mouse anti-human monoclonal Ki67 antibody, DAKO, 1:100), cleaved caspase-3 (apoptosis, rabbit polyclonal antibody, Cell Signaling, 1:100), CD31 (vascularization, mouse anti-human monoclonal antibody, DAKO, 1:20), and αSMA (smooth muscle actin, pericytes, mouse anti-human monoclonal, Chemicon, 1:500). Measurements of the tumor slide were performed in four random fields at ×40 magnification at the tumor invasive front. Ki67-positive and caspase-3-positive tumor cells were counted per high-power field (hpf), and averages were calculated. CD31-positive stained vessels and αSMA-positive areas were measured using ImageJ software and expressed as pixels per hpf.

Immunohistochemistry (IHC) staining for CD44v6 was performed in 3 µm TMA sections, using a mouse monoclonal antibody (clone MA54, 1:400; Invitrogen, Carlsbad, CA, USA). The assay was carried out on an automated Ventana BenchMark ULTRAStaining System, using the OptiView DAB IHC Detection Kit (both from Roche/Ventana Medical Systems, Tucson, Arizona, USA). The percentage of tumor cells displaying membranous expression of CD44v6 was assessed, and cases were classified as “CD44v6_0”—no staining at the cell membrane; “CD44v6_1+”—membranous staining in up to 10% of tumor cells; “CD44v6_2+”—membranous staining in between 11% and 50% of tumor cells; “CD44v6_3+”—membranous staining in between 51% and 75% of tumor cells; and CD44v6_4+—membranous staining in over 75% of tumor cells (Figure 1A).