Stage-specific embryonic antigen-4 (SSEA-4) Antibody Staining Protocol for Immunocytochemistry


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.

Cell Preparation:

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.


1. Fixation

hMSCs on the multiwell plate were washed three times with PBS, and fixated with 4% para-formaldehyde for 30 min at room temperature (RT).

2. Blocking

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.

5. Counterstain:

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.