Publication protocol
Pathological sections were obtained from all patients to the best of our ability, and all pathological sections obtained were reviewed. For each patient, tissue blocks of lesions of the peritoneum, appendix, and ovary were selected for immunohistochemical staining. Three-micrometer sections of the paraffin-embedded tissue were deparaffinized, rehydrated in a graded series of alcohol and microwave-treated for 10 min in a citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked using 0.3% hydrogen peroxide. The tissues were processed in an automatic immunohistochemical staining machine using the standard protocols (Lab Vision Autostainer; Lab Vision Co., Fremont, CA, USA) with DAKO Real™ EnVision™ Detection System (K5007, DAKO). We used the the following primary antibodies: CK7 (clone OV.TL-12/30, Dako; dilution 1:15 000), CK20 (clone Ks20.8, Dako; dilution 1:2000), MUC-1 (clone Ma 695, Novocastra; dilution 1:200) , MUC-2 (clone ccp58, Novocastra; dilution 1:100), CA125 (clone OC125, Dako; dilution 1:500), ER (clone 6F11, Novocastra; dilution 1:100), and PR (clone 16, Novocastra; dilution 1:200). All antibodies were incubated for 1 h at room temperature. The sections were visualized with 3-3′-diaminobenzidine and tissues were counterstained with Mayer’s hematoxylin. We used colon mucinous carcinoma tissue as a positive control for MUC2 and CK20, ovarian mucinous carcinoma tissue as positive control for MUC1, CA125 and CK7, and endometrial carcinoma tissue as a positive control for ER and PR. The same tissues without labeling by primary antibody were used as negative controls. Reactions were interpreted as positive, based on the presence of cytoplasmic staining for MUC2, CK7 and CK20 or cytoplasmic and membranous staining for MUC1 and CA125. For descriptive purposes, the staining was scored semi-quantitatively based on the percentage of positive cells: 1, negative; 2, < 10%; 3, 10-50%; and 4, > 50%. For comparative purposes, scores of 2-4 were considered to be positive.
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