Cd44v6

Antibodies to EpCAM (1:100 dilution), CD133 (1:100 dilution), CD44V6 (1: 50 dilution), and ALDH1A1 (1:50 dilution) were purchased from Abcam (Abcam, Cambridge, United Kingdom). Antibody to CD44v8-10 (1:50 dilution) was purchased from Cosmo Bio (Cosmo Bio, Tokyo, Japan).

Immunohistochemical analysis was performed as described before [22 (link)]. The material was routinely fixed in 4 % formaldehyde solution and embedded in paraffin. After slicing into 4-μm-thick sections, the preparations were dewaxed in xylene and then rehydrated. Endogenous peroxidase activity was blocked by 3 % hydrogen peroxide in methanol for 30 min. After a short rinse with phosphate buffered saline (PBS), sections were pre-incubated with avidin-biotin (Vector Laboratories, Peterborough, UK; SP-2001) for 15 min to reduce non-specific background staining. The preparations were covered with normal goat serum for 20 min and then incubated with the primary antibodies (CD44, mouse monoclonal, Diagnostic Biosystems, Pleasanton, CA, dilution 1:2000; CD44v6, abcam, Cambridge, MA, dilution 1:1000; and VEGF, mouse monoclonal, Dako, Denmark, dilution 1:50) for 30 min. Then, the sections were washed with PBS, incubated with biotinylated goat anti-mouse immunoglobulin G (BioGenex, Germany) for 30 min and covered with peroxidase-conjugated streptavidin (Dako). The peroxidase reaction was allowed to proceed for 8 min, with 0.05 % 3,3-diaminobenzidine tetrahydrochloride solution as substrate. Slides were counterstained with hematoxylin. Negative controls were also performed by replacing the primary antibodies with mouse or goat ascites fluid (Sigma-Aldrich, St. Louis, MO).

Tissue specimens were fixed with 4% formalin. Paraffin Sects. (5 μm) were prepared and fixed. Antigen retrieval was performed by incubating the cells in buffered citrate for 15 min at 105 °C. The sections were blocked with 5% w/v bovine serum albumin for 30 min and then incubated with primary antibodies against CD44s (1:100; Abcam, Cambridge, UK), CD44v3 (8 μg/mL; R&D Systems), and CD44v6 (1:100; Abcam) overnight at 4 °C. The slides were then stained with horseradish peroxidase-conjugated secondary antibodies, followed by counterstaining with diaminobenzidine (Agilent Technologies, Santa Clara, CA, USA) and hematoxylin. Images were visualized using a microscope (Olympus Corporation, Tokyo, Japan) or captured using a panoramic scanner PANNORAMIC (3DHISTECH Ltd., Budapest, Hungary). Each specimen was assigned a score according to the intensity of staining (negative staining = 0; week staining = 1; moderate staining = 2, and strong staining = 3). To diminish the manual errors, two pathologists performed the scoring independently. CD44s, CD44v3, and CD44v6 were quantified using the formula: IHC score = Σ (% of immunostained cells × intensity of staining).