Envision horseradish peroxidase labeled polymer

All samples were fixed with 10% (v/v) formalin, embedded in paraffin, and cut into 4-μm-thick sections. Antigens were retrieved using the HistoVTOne solution (Nacalai Tesque, Kyoto, Japan). The sections were incubated overnight at 4°C with a primary antibody to ephrin-B2 (Atlas Antibodies AB, Stockholm, Sweden), followed by incubation with the EnVision horseradish peroxidase-labeled polymer (Dako, Glostrup, Denmark) for 30 min and development with 3, 3 -diaminobenzidine (DAB) solution for 5 min. The sections were counterstained with Mayer’s hematoxylin. Results were classified according to two parameters based on a previously described modified method [20 (link)]: extent of ephrin-B2 staining (score: 0, no staining; 1, <35% positive cells; 2, 35%–75% positive cells; 3, >75% positive cells) and staining intensity (score: 0, no staining; 1, 2, and 3: weak, moderate, and strong staining, respectively). By multiplying the staining extent with the intensity, the following IHC staining grades were obtained: grade 0, no staining; grade 1–2, weak staining (+1); grade 3–4, moderate staining (+2); grade 6–9, strong staining (+3). Finally, for statistical comparisons, specimens with no (0) or weak staining (+1) were classified as the low ephrin-B2 group, whereas those with moderate (+2) or strong (+3) staining were classified as the high ephrin-B2 group.

Tumor and adjacent benign mucosal tissues of a subset of 40 HNSCC patients stained for SULF2 were used for paired analysis of 6-O-sulfated HSPG using the phage display antibody HS3A8 as described previously (15 (link)). The HS3A8 antibody was provided by Dr. Van Kuppevelt, University of Nijmegen, The Netherlands. The Ki67 antigen was stained with anti-Ki67 monoclonal antibody (Thermo Scientific) and visualized using EnVision+ horseradish peroxidase labeled polymer (Dako) according to the manufacturer’s instructions in the same sample set to evaluate the tumor cell proliferation. Briefly, 5 µm sections from FFPE tissues were deparaffinized with xylenes and rehydrated through a graded alcohol series. Slides were treated with 3% hydrogen peroxide and 10% normal goat serum for 10 min each and exposed to primary antibodies for Ki67 (Thermo Scientific) overnight at room temperature. Slides were incubated with EnVision+ HRP labeled polymer for 30 min and DAB chromagen (Dako) for 5 min. Slides were counterstained with Hematoxylin (Fisher, Harris Modified Hematoxylin), blued in 1% ammonium hydroxide, dehydrated, and mounted with Acrymount. Ki-67 was scored as proportional number of Ki-67 stained cells (in %) and intensity score in tumor tissue.