The study was approved by the ethical committee of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, China. In addition, informed consent was obtained from all of the subjects involved. Human breast tumor tissue arrays were obtained from Sir Run Run Shaw Hospital. 314 female cases were recruited with ages ranging from 24 to 75. The IHC analysis staining of paraffin-embedded tissue sections was carried out using the Dako Envision system (Dako, Glostrup, Denmark) as previously described76 (link). Briefly, the sections were submerged in boiling 10 mmol/L sodium citrate (pH, 6.0) for 2 min in a pressure cooker. After being treated with 0.3% hydrogen peroxide for 10 min to block endogenous peroxidase, the sections were incubated with primary antibody overnight at 4 °C. After washing, the sections were incubated with biotin-labeled secondary immunoglobulin (Dako) for 40 min at room temperature, followed by incubation with 3, 3′-diaminobenzidine (Dako) at room temperature. Images were taken by using an Olympus camera and matched software. The following primary antibodies were used: rabbit-ASCT2 (Abcam, ab84903; diluted at 1:800), rabbit-SPOP (Affinit, DF12106; diluted at 1:800).
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