Immunohistochemical Analysis of EMT Markers

IHC was performed as described previously [19 (link), 20 (link)]. Briefly, tissue sections were deparaffinized and dehydrated, incubated with H₂O₂, subjected to high-pressure repair, and blocked with BSA. anti-Numb (Abcam, Cambridge, UK, dilution: 1:400), Snail1 (Proteintech, Chicago, IL, dilution: 1:200), Snail2 (Proteintech, dilution: 1:200), E-cad (Abcam, 1:1000), N-cad (Proteintech, dilution: 1:200), Vimentin (Proteintech, dilution: 1:500), and Ki67 (Proteintech, dilution: 1:500) overnight. Slices were covered with the secondary antibody, detected with DAB, co-stained with hematoxylin, and evaluated by two professional pathologists for the staining scores.