Human CRC tissues were blocked and subsequently incubated with antibodies overnight at 4 °C, followed by incubation with the HRP-conjugated secondary detection antibodies (Dako Cytomation, USA) at room temperature for 30 min. The calculation of IHC staining scores was the same as the methods in our previous report [18]. Briefly, the staining intensity was independently scored by 2 pathologists blinded to the clinical data (0, no color; 1, weak; 2, moderate; 3, strong), and the percentage of positive cells (0, < 5%; 1, 6–25%; 2, 26–50%; 3, 51–75%; and 4, 76–100%) was semi-quantitatively assessed. The final scores (0–12) were then calculated by multiplying these 2 values. For analysis of clinical parameters, patients were divided into 4 subgroups based on this final score (negative, 0–2; weak, 3–4; moderate, 5–8; and strong, 9–12).

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