BMSCs were fixed with 4% (g/mL) paraformaldehyde for 15 min. After being washed three times in PBS, the cells were stained by FITC-labeled phalloidin (Beyotime, China) and DAPI (Beyotime, China). BMSCs morphology was observed by confocal laser scanning microscopy (LSM800, Zeiss, Jena, Germany). The experiment was performed in triplicate.
RAW 264.7 cells cultured on the scaffolds were fixed with 4% (g/mL) paraformaldehyde for 15 min. After being washed three times in PBS, the cells were permeabilized with 1% Triton X-100 for 30 min and blocked with 0.1% bovine serum albumin (BSA) for 1 h. The cells were incubated with primary antibodies against iNOS (1:100, Abcam, ab15323) and CD206 (1:400, Abcam, ab64693) overnight at 4°C. Then cells were rinsed three times with PBS and incubated with the corresponding secondary antibodies (Alexa Fluor® 488- or 594-conjugated goat anti-rabbit or anti-mouse IgG; both 1:1000; both Abcam; cat. nos. ab150113 and ab150080, respectively). After rinsed three times with PBS, the cells were stained with 10 µg/mL DAPI for 5 min at 25°C. Images were captured using a laser scanning confocal microscope (×400 magnification; Zeiss GmbH; cat. no. LSM780). All experiments were performed in triplicate.
RAW 264.7 cells cultured on the scaffolds were fixed with 4% (g/mL) paraformaldehyde for 15 min. After being washed three times in PBS, the cells were permeabilized with 1% Triton X-100 for 30 min and blocked with 0.1% bovine serum albumin (BSA) for 1 h. The cells were incubated with primary antibodies against iNOS (1:100, Abcam, ab15323) and CD206 (1:400, Abcam, ab64693) overnight at 4°C. Then cells were rinsed three times with PBS and incubated with the corresponding secondary antibodies (Alexa Fluor® 488- or 594-conjugated goat anti-rabbit or anti-mouse IgG; both 1:1000; both Abcam; cat. nos. ab150113 and ab150080, respectively). After rinsed three times with PBS, the cells were stained with 10 µg/mL DAPI for 5 min at 25°C. Images were captured using a laser scanning confocal microscope (×400 magnification; Zeiss GmbH; cat. no. LSM780). All experiments were performed in triplicate.