General sp kit

Tissue slides with paraffin sections were deparaffinized by xylene and ethanol. Endogenous peroxidase activity was blocked with 3% H₂O₂ for 15 min at room temperature. For antigen retrieval, slides were heated in citrate buffer for 20 min. Nonspecific binding was blocked with 10% fetal bovine serum in PBS for 15 min at 37°C. The slides were incubated with SQLE (1:200 dilution; Cat#12544‐1‐AP; Proteintech, Rosemont, IL, USA), Ki67 (1:5000 dilution; Cat#ab15580; Abcam, Cambrige, CB2, UK) at 4°C overnight. The slides were incubated with biotin‐conjugated secondary antibody using general SP kit (Cat#SP‐9000; ZSGB‐BIO, Beijing, China) and stained with the DAB substrate (Cat#SP‐9000; ZSGB‐BIO, Beijing, China). The immunoreactivity of tested samples was scored for multiplying staining intensity (0‐3) and positive cell proportion (0‐4).

A total of 450 samples were fixed by formalin, embedded in paraffin, and used for TMA construction. Tissue slides with paraffin sections were deparaffinized by xylene and ethanol and used for IHC analyses. Endogenous peroxidase activity was blocked with 3% H₂O₂ for 15 minutes at room temperature. For antigen retrieval, slides were heated in citrate buffer for 20 minutes. Nonspecific binding was blocked with 10% FBS in PBS for 15 minutes at 37°C. The slides were incubated with FDFT1 (1:200 dilution; Cat# 13128‐1‐AP; Proteintech), Ki‐67 (1:5000 dilution; Cat# ab15580; Abcam) at 4°C overnight. The slides were incubated with biotin‐conjugated secondary Ab using a general SP kit (Cat# SP‐9000; ZSGB‐BIO) and stained with the DAB substrate (Cat# SP‐9000; ZSGB‐BIO). The immunoreactivity of tested samples was scored for multiplying staining intensity (0‐3) and positive cell proportion (1‐4).