Immunohistochemical Analysis of SMAD4, LC3, and γ-H2AX

IHC staining was performed as previously described(29 (link)). Briefly, tumor sections were deparaffinized, rehydrated, and treated with 10 mM citrate buffer (pH 6.0) at 95°C to retrieve antigens. After quenching endogenous peroxidase activity with H₂O₂ and blocking with 10% horse serum, the sections were incubated sequentially with the primary antibodies mouse anti-SMAD4 (1:100; Cell Signaling Technology Inc. CA, USA), rabbit anti-LC3 (1:100; Cell Signaling Technology Inc. CA, USA), rabbit anti-γ-H2AX (1:100; Cell Signaling Technology Inc. CA, USA), biotinylated secondary antibodies, and the Avidin-Biotin Complexes (ABC) reagent (Gene Tech. San Francisco, CA, USA). The immunostaining was visualized with 3.3-diaminobenzidine (Gene Tech. San Francisco, CA, USA). The sections were then counterstained with hematoxylin. Negative controls were performed in each case by replacing the primary antibody with PBS.