Ab187532

Immunohistochemical staining was performed using the polymer Envision Detection System and Dako EnVision kit (Dako, Glostrup, Denmark). Tissue sections (3–5 μm) were deparaffinized in xylene and rehydrated in graded alcohol. The slides were incubated for 10 minutes in 3% hydrogen peroxide to block endogenous peroxidase activity, followed by treatment with Dako Target Antigen Retrieval Solution (pH 6.0). The slides were incubated with an anti-DKK3 antibody (ab187532; Abcam, Cambridge, UK). The reaction was visualized by incubating the sections with diaminobenzidine for 15 minutes. The sections were stained with Mayer hematoxylin. Immunohistochemical scoring was performed by a pathologist using a graded scale from 0 to 3+, where 0 to 2 represented weak staining, 2+ to 3 represented moderate staining, and 3+ represented strong staining.

Cells were solubilized in cold radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). Protein concentration was determined using the BCA assay kit (Thermo Fisher Scientific, Inc.) and proteins (50 µg) were separated with 10% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Thermo Fisher Scientific, Inc.). The membrane was blocked with PBS containing 5% milk (Yili Group, Beijing, China) for 3 h at room temperature. Following 3 washes with PBS (Beyotime Institute of Biotechnology), the membrane was incubated with rabbit polyclonal anti-DKK3 antibody (1:100; ab187532; Abcam, Cambridge, MA, USA) or rabbit polyclonal anti-GAPDH antibody (1:50; ab37168; Abcam) at room temperature for 3 h. Following 3 washes with PBS, the membrane was incubated with goat anti-rabbit immunoglobulin G (1:5,000; ab6721; Abcam) at room temperature for 40 min. An ECL kit (Thermo Fisher Scientific, Inc.) was then used to perform enhanced chemiluminent detection. Relative protein expression was presented as the density ratio vs. GAPDH using Image-Pro Plus software 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).