Ab264262

After hydrated with PBS, boiled in citrate buffer to retrieve antigens and incubated with 5% normal goat serum, the sections were performed with the anti-Wnt3a (ab219412, Abcam) or anti-β-catenin (ab264262, Abcam) at 4°C overnight. Subsequently, the sections were incubated with a biotinylated secondary antibody for 1 ​h at room temperature. Finally, HRP-streptavidin was performed for 15 ​min to visualize the immune reaction. All immunohistochemistry images were captured by a transmitted light microscope (CX31; Olympus) and the positive area (%) was calculated by Image-Pro Plus software (Version 6.0.0; Media Cybernetics Inc) according to the published literature [11 (link)].

Before protein extraction, the humerus-supraspinatus samples were frozen in liquid nitrogen. Then we removed the muscle belly, kept the tendon (one millimeter in length) and the portion of the humeral head proximal to the growth plate near the tendon attachment. The weight of each sample was adjusted to be around 20 ​mg. Samples were collected and lysed in RIPA lysis buffer. The insoluble materials were sedimented by centrifugation, while the supernatants were collected for protein extraction. The protein concentration was determined by the BCA assay. Protein extracts were separated by SDS-PAGE and blotted onto polyvinylidene fluoride membranes (Immobilon P, Millipore, Billerica, USA). After being blocked with 5% non-fat milk, membranes were incubated with a specific primary anti-beta catenin antibody (ab264262, Abcam) at 4°C overnight and with the horseradish peroxidase-conjugated secondary antibodies at 37°C for 1 ​h. Anti-β-actin and all the secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Finally, protein bands were visualized using an enhanced chemiluminescence reagent (Thermo Fisher Scientific, Waltham, USA). The reactions were detected by enhanced chemiluminescence assay using ChemiDoc XRS Plus luminescent image analyzer (Bio-Rad, Hercules, CA, USA).