Immunofluorescence Staining of Stem Cells

Immunofluorescence staining was performed as described [33 (link), 83 (link), 84 (link)]. Briefly, exponentially growing cells were fixed with 4% paraformaldehyde (PFA) for 10 min, washed with PBS and permeabilized with 1% NP-40 for 10min at room temperature. After being blocked with 10% donkey serum (Jackson Immuno-Research Laboratories, West Grove, PA) for 1h at room temperature, cells were incubated with various primary antibodies, including CD29, CD73, BMPRII, CD90, CD117/c-kit, CD105/endoglin, or BMPR-II antibody (all from Santa Cruz Biotechnology) for 1h at room temperature. Cells were washed with PBS and incubated with FITC-labeled secondary antibodies (Jackson ImmunoResearch Laboratories) for 30 min. DAPI (Invitrogen) was used to visualize nuclei. Stains were examined under a fluorescence microscope. Negative control cells were performed under the same conditions without primary antibodies. Representative images from at least three independent staining experiments are shown.