Paraffin-embedded slides of HCC tissue were heated at 60 °C for 1 h. Slides hydrated with xylene (Sigma-Aldrich, St. Louis, MO, USA) and gradient alcohol (Sigma-Aldrich, St. Louis, MO, USA) were dewaxed. For antigen extraction, slides were put into diluted potassium citrate (Sigma, Shanghai, China) solution and microwave- heated at 90 °C for 10 min. After cooling at room temperature, slides were rinsed with phosphate buffered saline (Mechanistic) (Sigma-Aldrich, St. Louis, MO, USA) for 3 times (5 min each time). Then, 3% H2O2 (Sigma-Aldrich, St. Louis, MO, USA) were added at room temperature. Subsequently, slides were blocked with 5% goat serum (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 20 ~ 30 min, followed by incubation with primary antibodies (MST, MST2, LATS1, LATS2, YAP1, TAZ, TEAD1, TEAD2, TEDA3, TEAD4, GAPDH) at 4 °C overnight. All antibodies were rabbit resource, diluted at 1:200, and purchased from Abcam (Cambridge, MA, USA). After washed with PBS (Sigma-Aldrich, St. Louis, MO, USA) for three times, slices were incubated with a goat-anti rabbit secondary antibody (Thermo Fisher Scientific, Shanghai, China) at room temperature for 1 h and then visualized using the DAB method (ZSGB-BIO, Beijing, China) according to manufacturer’s protocol. Subsequently, slices were stained with hematoxylin (Nanjing Jiancheng, Nanjing, Jiangsu, China) according to manufacturer’s protocols to visualize nuclei. For each sample, three fields of view were selected at random. Positive cells were counted.

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