Immunohistochemical Analysis of IL-8 in Colorectal Cancer

Immunohistochemistry analysis was conducted as previously described [22 (link)]. An EnVision™ III Detection System (GK500705; Gene Tech, Shanghai, China) was used during the process. The primary antibody included anti-IL-8 (Abcam). The images were captured using a Leica microscope.
We conducted the immunohistochemical (IHC) staining with the IL-8 antibody in a tumor microarray (TMA), which included 71 human colorectal cancer tissues. Additional file 1: Table S1 shows the patient characteristics. Two blinded independent pathologists, who had no knowledge of patient data and tumor characteristic, evaluated the IHC staining. The IL-8 staining was scored by two variables, one was the percentage of cells with stained cells (none = 0, 1–40% = 1, 41–75% = 2, and > 75% = 3), and the other was the intensity of the staining (none = 0, low = 1, moderate = 2, and strong = 3). The product of the two variables was considered as the immunoreactivity score (IS). Finally, the patients were divided into IL-8-low (IS< 2) and IL-8-high (IS ≥2) groups by IS values.