The EZH2 and pCHK1 (Ser345) expression levels were examined in 44 paraffin-embedded EOC tissues. The clinical and pathological characteristics of the patients from whom the tissues were collected are summarized in Table S6. Paraffin-embedded xenograft samples were used for the immunohistochemical staining of EZH2, Ki-67, ALDH1, E-CADHERIN and VIMENTIN. Endogenous peroxidase activity was blocked by treatment with 3% hydrogen peroxide in PBS for 30 min. The specimens were rinsed in PBS. The sections were incubated with primary antibodies (Table S3) overnight at 4 °C. The bound antibody was detected with a secondary biotinylated antibody for 30 min at room temperature and visualized using diaminobenzidine as a chromogenic substrate. The sections were then counterstained with hematoxylin. Positive expression was defined as brown-yellow granules in the cytoplasm or nucleus. The stained sections were evaluated and scored by two independent pathologists blinded to clinical and pathological data, based on both the percentage of positive cells (0, <10%; 1, 10%-20%; 2, 21%-50%; and 3, >50%) and the staining intensity (0, negative; 1, faint; 2, moderate; and 3, strong). Their product formula represents the final integral of each sample. Only cells associated with tumor islets were scored, and stromal and vascular staining was not evaluated. An immunohistochemistry (IHC) score less than 3 indicated low expression of EZH2 or pCHK1, while a score of 3 or greater indicated high expression. The controls used were isotype-matched IgGs.

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