Alexa fluor 568 red fluorescence conjugated goat anti rabbit

For immunofluorescence detections BioR/hGMSCs were fixed using 4% paraformaldehyde diluted in 0.1 M PBS (Lonza). After the fixation step, cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min, followed by blocking with 5% skimmed milk in PBS for 30 min.^{28 (link)} Primary antibodies used for immunofluorescence were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA, USA). COL1A1 (1:200, Santa Cruz Biotechnology), RUNX2 (1:100, Santa Cruz Biotechnology), BMP2/4 (1:200, Santa Cruz Biotechnology) and OPN (1:200, Santa Cruz Biotechnology) were used as primary antibodies. Then cells were incubated by Alexa Fluor 568 red fluorescence conjugated goat anti-rabbit as secondary antibodies (1:200, Molecular Probes, Invitrogen, Eugene, OR, USA). Alexa Fluor 488 phalloidin green fluorescence conjugate (1:400, Molecular Probes) has been used to mark the cytoskeleton actin. After immunofluorescence labelling cells were washed and incubated with TOPRO (1:200, Molecular Probes) for 1 h at 37°C.^{26 (link)} Samples were observed under Zeiss LSM800 confocal system (Zeiss, Jena, Germany). All the experiments were performed in triplicate.