Wnt10b antibody orb97574

The third generation of DPCs was plated on cover glass (WHB, China) in 6-well plates for culturing 4 days. Removed the basal medium and rinsed for three times using phosphate buffer solution (PBS). And then fixed with 4% paraformaldehyde (Solarbio, #P1110, China) for 30 min in room temperature and washed with PBS for three times, permeabilized with 0.5% Trixton X-100 for 15 min in room temperature and washed with PBS for three times, blocked with goat serum (BOSTER, #12C09A, China) for 30 min and incubated with α-SMA antibody (BOSTER, #BM0002, China) /or Vimentin antibody (BOSTER, #BM0135, China) /or Wnt10b antibody (orb97574, biorbyt, U.S.A.) overnight at 4°C in wet box [18–20 (link)]. For immunofluorescence staining, we used SABC-FITC SP kit (BOSTER, #SA1062, China). The DPCs were incubated with goat anti mouse IgG secondary antibody for 30 min at 37°C and then added SABC-FITC for incubating at 37°C in darkness for 30 min after washing with PBS. Counter-staining with DAPI and mounting with anti-fluorescence quenching agent (Beyotime, #P0128, China). The fluorescence signals were observed by using a fluorescence microscope (Nikon ECLIPSE 80i, Japan).

Total protein of DPCs was extracted with the cell active protein extraction kit (Sangon Biotech, #C500022, China). And the protein concentration was measured with a BCA Protein Assay Kit (Beyotime, China). For the Western blot analysis, equal protein amounts (40 µg per well) were denatured for 10 min at 100°C and separated with 12% gel electrophoresis. The protein was transferred onto a polyvinylidene fluoride (PVDF) microporous membrane at 200 mA and 4°C for 2 h. After 1 h of blocking in block solution (Beyotime, China) at room temperature, membranes were incubated at 4°C overnight in the following primary antibodies: Wnt10b antibody (orb97574, biorbyt, U.S.A.); β-catenin antibody (Cat. #06-734, Millipore, U.S.A.); Tubulin antibody (Beyotime, #AT819, China). The blots were then soaked with anti-mouse IgG-conjugated horseradish peroxidase (Beyotime, China) for 4 h at 4°C, and the protein bands were detected by Super Signal West Femto Maximum Sensitivity Substrate (Thermoscientific) or ECL (Beyotime, China). The results were visualized by exposing the blots to X-RAY film (Kodak). The films were scanned by a scanner (HP ScanJet 6100C) and signal intensity (Quantification) was conducted using ImageJ 1.43 software (National Institutes of Health, Bethesda, MD).