Dario Siniscalco and Umberto Galderisi
Second University of Naples, Italy
Stage-specific embryonic antigen-4 (SSEA-4, MC-813), a glycolipid carbohydrate epitope, is expressed on the surface of human teratocarcinoma stem cells (EC), human embryonic germ cells (EG) and human embryonic stem cells (ES); so that, it is an optimal marker to establish a difference between human and murine stem cells. It is used to characterize pluripotent stem cells. Mouse pluripotent stem cells are not recognised by anti-SSEA-4 antibodies but do express the antigen upon differentiation.
Human bone marrow mesenchymal stem cells (hMSCs) were grown in 10% FBS alpha-minimum essential medium (α-MEM), supplied with basic fibroblast growth factor (bFGF) on a coverglass placed in a 6 well tissue culture plate.
hMSCs on the multiwell plate were washed three times with PBS, and fixated with 4% para-formaldehyde for 30 min at room temperature (RT).
After washing 3 times with PBS, non-specific antibody binding was inhibited by incubation for 30 min in blocking solution (1% BSA in PBS).
3. Primary Antibody:
Primary antibodies were diluted in PBS blocking buffer and cells were incubated overnight at 4°C in primary antibodies to mouse anti-SSEA4 (1:100; Developmental Studies Hybridoma Bank, University of Iowa, IA).
4. Secondary Antibody and Detection:
HRP-labeled secondary antibodies (1:200; Jakson Immunoresearch Laboratories, West Grove, PA) specific to the IgG species used as a primary antibody and fluorescein labeled tyramide amplification (TSA; Perkin Elmer Life Science Products, Boston, MA) were used to locate the specific antigens.
After being washed three times with PBS, cells were counterstained with bisbenzimide (Hoechst 33258) and mounted with PBS:glycerol (90%).
Fluorescently labelled cells were viewed with a Zeiss Axioskop (Zeiss, Germany) epifluorescence microscope and the images were captured with a Zeiss Axiocam and Axiovison software.
Figure: SSEA4 immunostaining. SSEA4 immunostaining in human mesenchymal stem cells.