2.4. Immunohistochemistry (IHC)

IHC assays were performed on TMA slides after they were deparaffinized with xylene and rehydrated with a descending ethanol series. Antigen retrieval was conducted by high-pressure boiling in a citrate pretreatment solution to inhibit nonspecific antibody binding. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide, and nonspecific antibody binding was blocked with goat serum. The TMA slides were then incubated overnight in a humidified chamber at 4∘C with the following primary antibodies: a monoclonal antibody against FHIT (1:200, ProteinTech) or a monoclonal antibody against C-MYC (1:200, ProteinTech). An HRP Detection System was used to label reagents via incubation with a secondary antibody for 30 minutes at room temperature and incubation with DAB for two minutes. Finally, the slides were counterstained with hematoxylin, dehydrated through an ascending ethanol series, cleared with xylene, and mounted. Staining intensity was classified as negative, weak, moderate, or strong.