Quantifying Stem Cell Proliferation Using Ki67 Staining

Ki67 is a nuclear protein that is expressed in G1, S, G2, and early mitosis cells, but not in G0-phase cells^{72 (link)}; usually, the ratio of Ki67-positive cells can be used to assess the proliferation ability of SSCs^{73 (link)}. For staining of cultured SSCs, cells were fixed in 4% PFA for 10 min at room temperature. After the cells were washed in PBS, they were blocked in 10% donkey serum for 1 h at 37 °C and then incubated overnight at 4 °C with primary antibodies targeting Pax7 (1:100, no. AB528428, Developmental Studies Hybridoma Bank), MyoD (1:200, no. PA5-23078, Invitrogen, Carlsbad, CA, USA), and Ki67 (1:100, no. ab15580, Abcam, Cambridge, MA, USA). After three washes in PBS, the cells were incubated with secondary antibodies conjugated with Alexa Fluor Plus 488 (1:300, no. A32766, Invitrogen), Alexa Fluor 555 (1:400, no. A-31572, Invitrogen), and Alexa Fluor 488 (1:300, no. A-21206, Invitrogen) for 1 h at 37 °C. Finally, the cells were washed three times in PBS and stained with DAPI (no. C1005, Beyotime, Shanghai, China). RNA fluorescence in situ hybridization was performed with a Cy5-labeled (CAG)10 DNA probe (GenePharma, Shanghai, China), as described previously^{33 (link)}. Images were captured with a Nikon AL90 laser confocal scanning microscope (A1+R, Nikon, Tokyo, Japan) and analyzed with ImageJ software.