Dab substrate

Staining of formalin-fixed, paraffin-embedded breast cancer samples (139 cases that have received chemotherapy) was performed as previously described (Ghebeh et al, 2006 (link)). Briefly, after deparaffinization and rehydration, antigen retrieval (Dako) was used before blocking endogenous peroxidase for 15 min with 3% hydrogen peroxide (Sigma) in methanol. Sections were then blocked with 10% goat serum (Sigma) for 60 min, followed by the addition of a primary anti-human fascin antibody (1/200) overnight at 4 °C. After washing, sections were incubated with labelled Polymer (EnVision⁺ anti-mouse) HRP detection kit (Dako) for 30 min at room temperature. The HRP was detected using DAB substrate (Novocastra, Buffalo Grove, IL, USA) for 4 min and the sections were counterstained for 1 min with Harris hematoxylin (Acros Organics, Pittsburgh, PA, USA). Slides were washed with distilled water, dried and cover-slipped using permanent mount (Novocastra). The intensity of staining and the percentages of fascin-positive cells were evaluated by two independent pathologists who had no prior knowledge of patient details.

Formalin‐fixed, paraffin‐embedded breast cancer sections of 137 patients were deparaffinized and rehydrated as previously described.9 Antigen retrieval was done in citrate solution pH 6 (Dako) for 10 min using pressure cooker. Sections were incubated with 3% hydrogen peroxide (Sigma) in methanol to block endogenous peroxidase followed by 60 min incubation with 10% goat serum (Sigma). Sections were then incubated with anti‐ITGB1 antibody for overnight at 4^οC. The slides were washed before incubation with labeled Polymer (EnVision +) HRP detection kit (Dako) followed by DAB substrate (Novocastra). The sections were counterstained with instant hematoxylin (Shandon) and the intensity of staining and the percentages of ITGB1 positive cells were quantified at 5 to 10 increments by an anatomical pathologist who had no prior knowledge of patient details. Assessment of fascin expression on these patients has been previously described.9