Dylight 488 conjugated anti rabbit igg antibodies

Three Ф 20 mm titanium plates were taken from each group and placed into a 12-well plate. rBMSCs were inoculated on the titanium plates with a density of 2 × 10⁴/well. After incubating for 24 h, 48 h, and 72 h, respectively, cell proliferation was measured by cell staining and CCK-8 kit (Dojindo Molecular Technologies, Japan) according to the method previously described [33 (link)]. All these experiments were conducted in triplicates.
In addition, the expression of the marker of proliferation Ki-67 (Mki67) marker at each time point was observed by immunofluorescence assay. Cells on titanium discs were fixed in 4% paraformaldehyde (Sigma, USA) for 15 min and blocked with 10% goat serum (Solarbio, China) for 30 min. Subsequently, they were incubated with the primary antibody of Mki67 (Abclonal, China) overnight at 4°C. After rinsing, the cells were further incubated with DyLight 488-conjugated anti-rabbit IgG antibodies (Abcam, U.K.) for 1 h at ambient temperature. Finally, cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, China) for 15 min. Figures were then acquired using a fluorescence microscope (Nikon Eclipse TE300, Japan).

Three Ф 32 mm titanium plates were taken from each group and placed into a 6-well plate. rBMSCs were inoculated on the titanium plates with a density of 6 × 10⁴/well. After incubating for 14 d in osteogenic medium, immunofluorescence staining was carried out to detect the expressions of osteocalcin (OCN). Cells on titanium discs were fixed in 4% paraformaldehyde (Sigma, USA) for 15 min and blocked with 10% goat serum (Solarbio, China) for 1 h. Subsequently, they were incubated with the primary antibody of OCN (Santa, USA) overnight at 4°C. After rinsing, the cells were further incubated with DyLight 488-conjugated anti-rabbit IgG antibodies (Abcam, U.K.) for 1 h at ambient temperature. Finally, they were stained with rhodamine-phalloidin (Cytoskeleton, USA) for 1 h, and cell nuclei were stained with DAPI (Beyotime, China) for 10 min. Figures were then acquired using a fluorescence microscope (Nikon Eclipse TE300, Japan).