β catenin

Immunohistochemical (IHC) staining was carried out as previously described [24 (link)]. Five micrometer sections of paraffin-embedded human EOCs, borderline tumors, benign tumors, and normal ovarian tissues were prepared for staining. After dewaxing, the sections were rehydrated, then antigen retrieval and endogenous peroxidase blocking were performed. The slides were incubated with monoclonal antibodies (PPA1: Sigma, St. Louis MO, 1:250; E-cadherin: BD Bioscience, San Jose CA, 1:200; Vimentin: Cell signaling Technology, Danvers, MA, 1:200) overnight at 4°C. The sections were then incubated with biotinylated anti-mouse (PPA1, E-cadherin) or anti-rabbit (Vimentin, β-catenin) antibody (vector laboratories, Burlingame, CA) followed by HRP-streptavidin. Antigens were detected with peroxidase substrate and counterstained with hematoxylin. Scoring was based on the intensity and proportion of positively stained cells. The intensity of positive staining was scored from 0 to 3 as follows: 0 (none), 1 (low), 2 (moderate), and 3 (high). The proportion was scored from 0 to 4 as follows: <10% was scored as 0; 10–25% was scored as 1; 26–50% was scored as 2; 51–75% was scored as 3 and >75% was scored as 4. These two values were multiplied to produce the total score. A score <2 was considered PPA1-negative, 2–5 was considered PPA1 weak staining, and 6-12 was considered PPA1 strong staining.