Alexa fluor 594 antibodies

Representative proteins (Runx2 and OPN) indicating osteogenic differentiation were evaluated in the BMSC, BMSC/BMP-4, BMSC/RAW, and BMSC/RAW/BMP-4 groups by fluorescence staining. Briefly, after culture of cells in conditioned medium for 12 days, scaffolds were fixed with 4% paraformaldehyde (Biosharp) for 15 min, permeabilized with 0.1% Triton-X for 30 min, and blocked with 5% BSA for 1 h. Samples were then incubated with primary polyclonal IgG rabbit anti-rat antibodies targeting Runx2 (1:500; Abcam) and OPN (1:200; Abcam) overnight at 4 °C and then with secondary goat anti-rabbit Alexa Fluor 594 antibodies (1:200; Abcam) for detection of Runx2 or secondary goat anti-rabbit Alexa Fluor 488 antibodies (1:500; Abcam) for detection of OPN for 2 h at room temperature. The cytoskeleton was stained with Alexa Fluor 488 for detection of Runx2 or Alexa Fluor 594 phalloidin (ThermoFisher) for detection of OPN for 2 h at room temperature. Nuclei were stained with DAPI for 5 min. A fluorescence confocal microscope (Leica, Germany) was used to acquire representative images. Image Pro Plus 6.0 software (NIH) was used to quantitatively analyze the fluorescence intensity of Runx2 and OPN in five different views for three independent scaffolds.

The polarization of RAW264.7 cells in the RAW and RAW/BMP-4 groups was assessed by fluorescence staining to evaluate the expression levels of CCR7 (red, M1 marker) and CD206 (green, M2 marker). Briefly, after incubation in high-glucose DMEM for 2 and 5 days, scaffolds were fixed with 4% paraformaldehyde (Biosharp, Hefei, China) for 15 min, permeabilized with 0.1% Triton-X for 30 min, and blocked using 5% bovine serum albumin (BSA) for 1 h. Samples were incubated with M1 primary polyclonal IgG rabbit anti-mouse antibodies for CCR7 (1:100; Abcam) overnight at 4 °C and then with secondary goat anti-rabbit Alexa Fluor 594 antibodies (1:500; Abcam) for 2 h at room temperature. Next, samples were incubated with M2 primary polyclonal IgG mouse anti-mouse antibodies for CD206 (1:50; Abcam) overnight at 4 °C and then with secondary goat anti-mouse Alexa Fluor 488 antibodies (1:500; Abcam) for 2 h at room temperature. Finally, the nuclei were stained blue with 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) for 5 min and observed with a fluorescence confocal microscope (Leica, Germany). Image Pro Plus 6.0 software (NIH) was used to quantitatively analyze the fluorescence intensities of CCR7 and CD206 for five different view regions from three independent scaffolds.