Dario Siniscalco and Umberto Galderisi
Second
University of Naples, Italy
Description
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.
Procedure:
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%).
Observation:
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.
Results:
Figure: SSEA4 immunostaining. SSEA4 immunostaining in human
mesenchymal stem cells.
References: