Anti β catenin

The paraffin-embedded tumor tissue sections were deparaffinized and rehydrated for IHC, and the antigen was retrieved with high pressure in 0.01 M sodium citrate buffer solution. After incubating with the primary and secondary antibodies, the sections were incubated with diaminobenzidine and counterstained with hematoxylin (Solarbio, China). Images were taken by a microscope with 200× magnification (Olympus, Japan). Primary antibody for IHC: anti-Twist1 (Bioss, USA), anti-β-catenin (Bioss, USA).

A standard IHC technique was performed to determine the expression of TRIM36 and β-catenin in tissue samples. After deparaffinization and rehydration, antigen retrieval was performed with 0.01 M citrate buffer (pH 6.0) in a microwave for 15 min. Following the inactivation of endogenous peroxidases and the blockage of nonspecific antigens, the sections were incubated with anti-TRIM36 (Abcam, Cambridge, MA, USA) or anti-β-catenin (Bioss Inc., Woburn, Massachusetts, USA) overnight at 4°C. After washing, the slides were probed with horseradish peroxidase- (HRP-) conjugated secondary antibody for 1 h at room temperature, treated with the 3,3-diaminobenzidine (DAB) solution, and counterstained with hematoxylin. The slides were reviewed independently by two pathologists. The immunoreactive score (IS) was assessed by using the following formula: IS = percentage (0, no positive cells; 1, 1%–10%; 2, 11%–50%positive; 3, >50%positive) × staining intensity (0, absent; 1, weak; 2, moderate; 3, strong). The target protein was classified as high expression when IS ranged from 3 to 9, otherwise classified as low expression.