Paraffin wax was used for embedding the sample tissues. Deparaffinisation of paraffin sections (thickness = 5 μm) was performed using xylene and graded alcohols. Antigen retrieval was performed using microwave treatment with a citric acid solution (pH 6.0) for 20 min. Samples were then cooled at room temperature and subsequently treated with 3% hydrogen peroxide for 10 min, also at room temperature, to quench endogenous peroxidase activity. Nonspecific binding was mitigated by incubating with goat serum for 1 h. Subsequently, staining was conducted using haematoxylin-eosin or overnight at 4 °C with the following primary antibody: Ki67 (9449, CST). Staining was examined using HRP Envision Systems (Dako, Shanghai, China).
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