Anti cd44

Tumor samples were distributed in 15 TMAs using 4 mm tissue cores at the Experimental Pathology Laboratory of Pontifical Catholic University of Paraná (PUCPR). From each donor block was extracted one or two cylinders 4 mm in diameter and deposited in the receiver blocks, previously prepared. In each TMA, a sample of normal breast tissue was included as an internal control. Thereafter, 4 μm tissue sections from the TMA blocks were transferred to electrically charged Star Frost® (Braunschweig, Germany) slides and incubated with primary antibodies [anti-CD44 (anti-human mouse monoclonal, clone DF1485, dilution 1/40, Novocastra, Newcastle, UK), anti-CD44v6 (antihuman mouse monoclonal, clone VFF-7, dilution 1/100, Novocastra, Newcastle, UK), anti-CD24 (anti-human rabbit polyclonal, dilution 1/200, Abbiotec, San Diego, CA, USA), and anti-ALDH1 (anti-human rabbit monoclonal, clone E-P1932y, dilution 1/100, Epitomics, Cambridge, Massachusetts, USA)] for 12 h in a humidified chamber at 2−8°C. An Advance Dako (Caripenteria, CA, USA) secondary antibody was incubated with the slides for 30 min at 2−8°C. The reactions were developed using a DAB chromogen-substrate solution (Dako). Harris hematoxylin was used for counterstaining. Positive and negative (incubated without primary antibody) controls were run in parallel with all reactions.

Immunohistochemistry was performed on 4-μm TMA sections using a Ventana BenchMark XT Staining systems (Leica Microsystems, Wetzlar, Germany) according to the manufacturer’s instructions. The primary antibodies used were anti-EPHB2 (1:700, R&D Systems, Minneapolis, MN, USA), anti-CD44 (1:100, Novocastra Laboratories Ltd., Newcastle upon Tyne, UK), and anti-β-catenin (1:800, 17C2, Novocastra Laboratories). The expression of EPHB2 and CD44 was determined by examining the tumor cell membrane. For each tumor, the intensity and percentage of tumor cells expressing EPHB2 (n = 567) or CD44 (n = 87) were evaluated. Histoscores (H-scores) were calculated by multiplying the intensity score (0, negative; 1, weak; 2, moderate; and 3, strong) and percentage of positive tumor cells (range, 0 to 100), ranging from 0 to 300. For statistical analyses for EPHB2, we used a cutoff of 40 on the basis of the distribution of the H-scores (median value, 40). CRCs with H-score of 40 or lower were classified as negative, while cases with H-score higher than 40 were classified as positive. β-Catenin staining was considered positive when more than 10% of tumor cell nuclei were strongly stained.