Immunohistochemical Analysis of β-Catenin

This experiment was detected according to Wu et al.'s method [24 (link)], briefly described in the following: firstly, the tissues were formalin-fixed and then paraffin-embedded. The sections from the tissues were deparaffinized and dehydrated. They were boiled for 10 min in 0.01 M citrate buffer and incubated with 0.3% hydrogen peroxide (H₂O₂) in methanol for 15 min to block endogenous peroxidase. The sections were then incubated with the anti-β-catenin polyclonal antibody (1 : 300 dilution; sc-7199, Santa Cruz) overnight at 4°C, following incubation with secondary antibody tagged with the peroxidase enzyme (SP-9000, Zhongshan Golden Bridge, China) for 30 min at room temperature, and were visualized with 0.05% 3,3-diamino-benzidine tetrachloride (DAB) till the desired brown reaction product was obtained. The sections were finally counterstained with hematoxylin. Control sections were performed using phosphate-buffered solution (PBS) without a primary antibody. All the slides were observed under a Nikon E400 Light microscope, and representative images were taken.