Tissue specimens were fixed with 10% formalin, embedded in paraffin, and cut into 5μm-thick sections. After cleaned in xylene and rehydrated through an ethanol gradient, the sections were treated with 3% hydrogen peroxide to quench endogenous peroxidases, and then boiled in 10mM citrate buffer (pH 6) for antigen retrieval. The processed sections were then blocked with 10% goat serum for 30 min, and incubated overnight with 1:200 diluted polyclonal anti-human AKR1B10 (BOSTER, Wuhan, China) or anti-human FGF1 (BOSTER, Wuhan, China) at 4°C. Color was developed using a tissue staining kit (Zhongshan Biotechnology, Beijing, China). The AKR1B10 or FGF1 staining scores were evaluated in five random fields per slide by two pathologists YuHong Wang (The First Affiliated Hospital of Soochow University) and Zheng Zhi (The Soochow University) in a blinded manner as previously described [24 (link)]. The percentage of positively stained cells was scored as follows: 0 - 0-5%; 1 - 6-25%; 2 - 26-50%; 3 - 51-75%; 4 - >75%. The staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). The final score was the average of the percentage score multiplied by intensity score, and graded as follows: – (0), + (1-4), ++ (5-8) and +++ (9-12). Samples with final scores ++ or +++ were graded as positive, and – or + as negative.
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