Alexa fluor 647 conjugated goat anti rabbit igg

To investigate the expression of Wnt signaling pathway-related protein in rBMSCs, a confocal laser scanning microscope was used to acquire immunofluorescence images. Briefly, rBMSCs were seeded at a density of 1 × 10⁴ cells per well on the L-M/CS-8 and CS in 24-well culture plates and cultured for 3 days. Four percent paraformaldehyde was used to fix rBMSCs. The rBMSCs were permeabilized with 0.1% Triton X-100 and blocked with 1% BSA (Bovine albumin) solution at 37°C for 1 h. Subsequently, cellular specimens were incubated with β-catenin primary antibody (Santa Cruz Biotechnology, USA) at 4°C overnight. After washing thrice in PBS buffer, the cells were incubated with Alexa Fluor 647-conjugated goat-anti-rabbit IgG (Cell Signaling Technology, USA) for 1 h at room temperature. FITC-conjugated phalloidin (Molecular Probes, USA) was used for labeling the cytoskeleton for 20 min at room temperature. Cellular specimens were washed thrice in PBS and treated with 1 μg·ml⁻¹ of DAPI in PBS for 5 min to stain the cell nucleus. Immunofluorescence images were obtained using confocal microscope. To examine the effect of the Wnt signaling pathway in L-M/CS-8-stimulated β-catenin expression, cells were treated with the Wnt signaling pathway inhibitor Dickkopf-1 (DKK1, PeproTech, USA) in 0.5 μg·ml⁻¹ for 3 days. Immunofluorescence staining was performed by the same methods as described above.