2.13. Immunohistochemical Analysis in Tumor Xenografts

Immunohistochemistry (IHC) for Ki-67, p-Akt, and E-cadherin was performed using rabbit monoclonal antibodies. Tumor tissues were deparaffinized with xylene twice for 5 min and rehydrated and rinsed with tap water for 10 min. They were incubated at 4°C with primary antibodies overnight. Immunoreactivity was visualized with an avidin-biotin peroxidase reaction (PK-4001, Vectastain ABC kit). The peroxidase reaction was developed using a 3,3′-diaminobenzidine tetrahydrochloride (D-5905, Sigma). The sections were counterstained with hematoxylin before being mounted.