Ab15251

The tissues were cut into 4 μm-thick sections, followed by dewaxing and dehydration. Then the tissue sections were immunostained with the following diluted antibodies purchased from Abcam Inc.: rabbit monoclonal antibody to PTEN (ab32199, 1:1000), rabbit polyclonal antibody to Wnt (ab15251, 1:1000), and rabbit monoclonal antibody to β-catenin (ab32572, 1:1000). Next, the tissue sections were incubated with biotin-labeled secondary goat anti-rabbit IgG (ab6721, 1:1000, Abcam Inc.) for 30 min. After staining with 3,3′-diaminobenzidine (DAB; DA1010, Beijing Solarbio Science and Technology Co. Ltd., Beijing, China), the sections were dehydrated, cleared, and mounted. Finally, five high-power visual fields were randomly selected from each section and 100 cells were counted in each field under a light microscope (XSP-36, Boshida Optical Instrument Co. Ltd., Shenzhen, China). Positive cells <5% are negative, and positive cells ⩾5% are positive. The results were scored by two people independently.

After the in vivo tumor is prepared into the slide, immunohistochemistry staining was performed using immunohistochemical kit (ZSGB-BIO, Beijing, China) according to the manufacturer's protocol. And primary antibodies against β-catenin (Abcam, ab32572, Cambridge, England) and wnt1 (Abcam, ab15251) were used.

For western blot analysis, total protein was extracted from transfected cells using radioimmunoprecipitation assay (RIPA) buffer (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) and the supernatants were collected with centrifugation at 12,000 × g at 4°C for 20 min. Protein concentration was measured with a Bicinchoninic acid Protein Assay kit (Beyotime Institute of Biotechnology, Haimen, China). Subsequently, equal amounts of protein (30 µg) from each sample were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat milk for 1 h at room temperature and then incubated with primary antibodies overnight at 4°C. The specific primary antibodies were as follows: Anti-wnt (ab15251; 1:1,000; Abcam, Cambridge, UK), p-GSK3 (ab75745; 1:1,000; Abcam), GSK3 (ab32391; 1:1,000; Abcam), β-catenin (ab32572; 1:1,000; Abcam). Following the membranes were washed with TBST containing 0.2% Tween-20 three times and incubated with the horseradish peroxidase-conjugated anti-rabbit secondary antibody (cat. no. ab6721; 1:2,000; Abcam) at room temperature for 1 h. The blot was visualized by an odyssey infrared imaging system with ECL western blotting substrate kit (Amersham; GE Healthcare, Chicago, IL, USA). Expression of GAPDH was used as a loading control.