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Endoglin (CD105) Antibody Staining Protocol for Immunocytochemistry
Amalia Forte, Dario Siniscalco, and Umberto
Galderisi
Second
University of Naples, Italy
Description
Endoglin (CD105), an endothelial homodimeric membrane antigen
containing the tripeptide sequence RGD, is expressed on the
endothelial cells of capillaries, arterioles and venules, such as on
myelocytic leukaemia cells. Endoglin is a glycoprotein and it is
part of the transforming growth factor receptor complex that binds
several members of the TGFbeta superfamily. Endoglin is also a
marker to phenotypically characterize mesenchymal stem cells (MSCs).
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 glass-slide culture chamber.
Procedure:
1.
Fixation
hMSCs on the chamber were washed three times with PBS, and fixated
with 4% paraformaldehyde 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-endoglin (1:250; Chemicon/Millipore,
Billerica, MA).
4.
Secondary Antibody and Detection:
Fluorophore-labeled secondary antibodies (1:1000; Molecular Probes/Invitrogen,
Paisley, UK) specific to the IgG species used as a primary antibody
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%).
6.
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.
7.
Results:
References: