Tritc conjugated anti rabbit antibody

Aliquots of 5 × 10³ MSCs labeled with CFSE and 5 × 10³ MEFs (or 1 × 10⁴ MEFs alone) in serum-free DMEM culture media were cultured on the upper layer of Corning cell culture inserts with an 8.0 μm polycarbonate membrane. A DMEM culture medium with 10% FBS and the indicated cytokines or chemicals was added below the cell’s permeable membrane. Following an incubation period of 24 h at 37 °C and 5% CO₂, the cells that had migrated through the membrane were harvested. For immunofluorescence, cells were fixed with 4% paraformaldehyde (Sigma) for 20 min. Then, 10% bovine albumin (BSA) was used to block the cells for 20 min at room temperature. Afterwards, the cells were incubated with anti-α-Sma (Servicebio, GB111364) overnight at 4 °C. On the second day, the cells were incubated with the TRITC-conjugated anti-rabbit antibody (Abclonal, AS040) for one hour at room temperature. DAPI (Solarbio, C0060) was used to stain the nucleus. Images were taken using a Zessis Axio Scope A1 FL for FISH microscope. For crystal violet staining, cells were fixed with methanol for 20 min and 0.1% crystal violet was added. Photos were taken using an Olymbus IX50/70 microscope system.

BMP2 (R&D, Minnesota, USA, 355-BM-100), DKK1 (BioLegend, California, USA, 759602), 4′,6-diamidino-2-phenylindole (DAPI, Solarbio, Beijing, PRC, C0060), carboxyfluorescein succinimidyl ester (CFSE, eBioscience, California, USA, 65-0850-84) and gallocyanine (Selleck, Texas, USA, S5602) were used. The antibodies used in this study were monoclonal anti-YAP (Cell Signaling Technology, Boston, USA, 14074), anti-pYAP S127 (Cell Signaling Technology, Boston, USA, 13008), anti-actin alpha2, smooth muscle (anti-α-Sma, Servicebio, Wuhan, PRC, GB111364), anti-osteocalcin (anti-OCN, BioByt, Cambridge, UK, orb259644), tetramethylrhodamine isothiocyanate (TRITC)-conjugated anti-rabbit antibody (ABclonal, Wuhan, PRC, AS040), fluorescein 5-isothiocyanate (FITC)-conjugated anti-mouse antibody (ABclonal, Wuhan, PRC, AS001) and anti-β-actin (Sigma, St. Louis, USA, A1978). Tris-HCl, NaCl and other chemicals were purchased from Sigma.