Ab207298

The surgically resected sections of the highest nuclear grade were selected in each case, and FFPE specimens were prepared for immunohistochemical study. We used primary antibodies against the forkhead box protein M1 (FOXM1; 1:100; rabbit monoclonal, ab207298; Abcam), polo‐like kinase 1 (PLK1; 1:100; rabbit monoclonal, 4513; Cell Signaling Technology), and cyclin‐dependent kinase 1 (CDK1; 1:100; mouse monoclonal, MA5‐11472; Thermo Fisher Scientific).
Immunohistochemical staining was independently assessed by two pathologists. The nuclear staining intensity was classified into four categories: “0” for negative staining, “1” for weak staining, “2” for moderate staining, and “3” for strong staining. For FOXM1 and PLK1, we regarded tumor cells with a nuclear staining intensity ≥2 as positive and evaluated the labeling index in a hot spot (Figure S1B). A labeling index ≥3% was defined as indicative of a positive case for FOXM1, and the existence of any positive tumor cells was defined as a positive case for PLK1. On the other hand, for CDK1, the H‐score was calculated as previously described (Figure S1B).³⁰, ³¹ An H‐score ≥75 was defined as indicative of a positive case for CDK1. These cutoff values were determined by a time‐dependent receiver‐operating characteristic (ROC) curve, as described below.

The sections were deparaffinized in xylene and hydrated in a graded series of ethanol, then antigen retrieved by heat exposure in Tris/EDTA buffer (pH 9.0) for 15 min, and blocked for endogenous peroxidase activity in 3% hydrogen peroxide at room temperature for 10 min. Followed by blocking with 5% normal blocking serum, the sections were reacted with anti-CENPF (1:200 dilution, ab5) (28 (link)) or anti-FOXM1 (1:250 dilution, ab207298) (both from Abcam) (29 (link)) at 4°C overnight. After probing with HRP-conjugated secondary antibody, immunocomplexes were visualized using 3,3′-diaminobenzidine (DAB) (both from Long Island Biotech Co., Ltd.) and lightly counterstained with hematoxylin. The sections were reviewed and classified by two independent investigators as previously described (27 (link)). The percentage of positive stained cancer cells was scored as 0, negative; 1, 1–10%; 2, 11–50% positive; 3, >50% positive. The staining intensity was scored as 0, absent; 1, weak; 2, moderate; 3, strong. The immunoreactive score (IS) was calculated as follows: IS= percentage × staining intensity. The protein was considered to be highly expressed when IS was 3–9, otherwise to be lowly expressed.