Dako envision flex peroxidase blocking reagent

Formalin-fixed, paraffin-embedded (FFPE) tissues from 33 patients were subjected to a staining procedure with DAKO EnVision™ FLEX peroxidase blocking reagent (K8000; Dako, Hamburg, Germany) and antigen retrieval via pre-treatment with citrate buffer pH 6.0 (Abcam, Cambridge, UK) for 20 min. The slides were then incubated with primary antibodies to either E-cadherin (1:400, ab40772) or α-catenin (1:100, ab51032) (both from Abcam) for 120 min at room temperature. Following this, dextran polymer conjugated horseradish peroxidase and 3,3′-diamino-benzidine (DAB) chromogen was used for visualization and hematoxylin solution (Gill 3; Sigma-Aldrich, Munich, Germany) for counterstaining. Negative control slides in the absence of primary antibodies were included for each staining. Marker expression was evaluated semi-quantitatively by two independent investigators (F.R. and S.H.) without knowledge of the clinical characteristics. The percentage of positive tumor cells was assigned to one of the following categories: 0 (<5%), 1 (5–25%), 2 (26–50%), 3 (51–75%) and 4 (>75%). The intensity of immunostaining was scored as follows: 1⁺ (weak), 2⁺ (moderate) and 3⁺ (intense) and the percentages of positive tumor cells and staining intensity were then multiplied to produce an individual weighted score of 0–12.