IHC

BC tissues and ANBTs were fixed in 4% formalin solution for 24 h at room temperature, then embedded in paraffin and sectioned (5-µm-thick). Paraffin sections were heated at 55˚C for 2 h. Prior to immunostaining, slides were dewaxed in xylene and rehydrated in alcohol, and antigen retrieval was performed by microwaving the slides in citric saline (Wuhan Promoter Biological Co., Ltd.). The slides were subsequently incubated with 3% hydrogen peroxide to inhibit endogenous peroxidase activity for 15 min at room temperature and blocked with 3% goat serum (Sigma-Aldrich; Merck KGaA) for 10 min at 37˚C. For IHC staining, the slides were incubated with primary antibody against SULT1A2 (cat. no. HPA051051; 1:200; Sigma-Aldrich; Merck KGaA) overnight at 4˚C, and subsequently incubated with secondary antibody [GTVision I; 1:500; Gene Science and Technology (Shanghai) Co., Ltd.] for 1 h at room temperature. SULT1A2 expression was detected using a DAB detection system [Gene Science and Technology (Shanghai) Co., Ltd.]. The slides were visualized and captured at x400 magnification (Axio Imager. Z2 fluorescence microscope; ZEISS).

IHC staining was assessed using a semi-quantitative scoring method (15) by recording both the area of positive staining and the staining intensity. The area of positive staining was scored as follows: 0, 0%; 1, 1-25%; 2, 26-50%; 3, 51-75% and 4, >75%. The staining intensity was defined as follows: 0, no staining; 1, weak staining; 2, moderate staining and 3, strong staining. The immunoreactivity score (IHS) was calculated by multiplying the positive area score by the staining intensity score. An IHS <8 was classified into the low expression group, while an IHS ≥8 was classified into the high expression group.