IHC and Masson's Trichrome Staining

Paraffin embedded tissue specimens were sectioned, dewaxed, and rehydrated. Antigen retrieval was performed in 10 mM sodium citrate buffer (pH 6.0) at 95–100°C for 20 min. Endogenous peroxidase activity was blocked by 3% H₂O₂ for 30 min. Sections were then blocked in 5% BSA for 1 h and incubated with primary antibodies overnight. After extensive washing, the sections were incubated with secondary antibodies at room temperature for 1 h. DAB solution was applied and hematoxylin was used for counterstaining. The primary antibodies used in Immunochemistry were anti-YAP (CST, 1:200), anti-ANKRD1 (Santa Cruz, 1:100), and anti-CK19 (Abcam, 1:800). Collagen fiber deposition in tissue sections were visualized by Masson's trichrome staining. Staining results were quantified using Image-Pro Plus software. IOD/Area was used to quantify the protein expression in Immunohistochemistry (IOD: integrated option density; Area: total area of staining region).