Evaluating Pluripotency Markers in hMBSCs

Immunofluorescence experiments were carried out following our previously reported protocols [37 (link)]. Briefly, cells growing on the glass slide were fixed with 4% paraformaldehyde for 15 min and permeabilized using 0.25% Triton X-100 diluted in PBS for 10 min at room temperature. To block unspecific epitopes, cells were incubated with PBS containing 1% BSA and 0.1% Tween-20 for 1 h. To evaluated the pluripotency of hMBSCs, we used the following antibodies: rabbit anti-OCT₄ (5 μg/ml, Abcam, Nanchang, China), mouse anti-SSEA-4 (15 μg/ml, Abcam), and rabbit anti-Nanog (1 : 200, Abcam). The fixed cells were incubated overnight at 4°C with primary antibodies followed by incubation with secondary donkey anti-mouse or anti-rabbit antibodies conjugated to either Alexa Fluor 488 or Alexa Fluor 568 (Jackson, Nanchang, China). Nuclei were counterstained with DAPI (Thermo Fisher).