saponin/cytokine IHC

From:"C.M. vander Loos" <>

Dear Jim, Kim, John, Gayle and all other IHC-cytokine victims,

Our IHC IFNgamma study (2nd version) is still under J Histochem Cytochem
Editorial consideration. The abstract below is already published in: XIth
Int. Congr. of Histochem. Cytochem., Univ of York, UK, 3-8 Sept. 2000, pp.
I'll keep you informed!

Happy holidays


C.M. van der Loos, Dept. of Cardiovascular Pathology, Academical Medical
Center, Meibergdreef 9, NL-1105 AZ Amsterdam, The Netherlands

C.M. van der Loos, M.A. Houtkamp, O.J. de Boer, P. Teeling, A.C. van der
Wal and A.E. Becker
Dept. of Cardiovascular Pathology, Academical Medical Center, Meibergdreef
9, NL-1105 AZ Amsterdam, The Netherlands

Immunohistochemistry is a widely accepted tool to investigate the presence
and immunolocalization of cytokines in tissue sections at the protein
level. We have tested the specificity and reproducibility of
IFNg-immunohistochemistry on tissue sections with a large panel of
anti-IFNg antibodies.

Thirteen different commercially available anti-IFNg antibodies, including 7
advertized and/or regularly applied for
immunohistochemistry/-cytochemistry, were tested using a 3-step
streptavidin-biotin peroxidase technique and a 2-step immunofluorescence
(FACS) analysis. Immunoenzyme double staining was used to identify the
IFNg-positive cells.
Serial cryostat sections were used of human reactive hyperplastic tonsils,
rheumatoid synovium and inflammatory abdominal aorta aneurysms, known to
posses a prominent Th1-type immuneresponse. In vitro phorbol myristate
acetate/ionomycin stimulated T-cells served as positive control;
unstimulated cells served as negative control. Cultured T-cells were used
adhered to glass-slides (immunocytochemistry), in suspension (FACS), or
were snap-frozen and sectioned (immunohistochemistry).

1.	Immunocytochemistry and FACS analysis on stimulated cultured
T-cells showed positive staining results with 12 of 13 anti-IFNg
antibodies. However, immunohistochemistry of sectioned stimulated T-cells
was negative with all. Unstimulated cells were consistently negative.
2.	IFNg-immunohistochemical single- and double staining analysis of
the tissue sections showed huge variations in staining patterns, including
positivity for smooth muscle cells (n=8), endothelial cells (n=4),
extracellular matrix (n=4) and CD138+ plasma cells (n=12). Specific
staining of T-cells, as the sole positive staining, was not achieved with
any of the 13 antibodies.

IFNg-immunohistochemistry appears unreliable because of lack of specificity
to stain T-cells in situ. In fact, depending on the type of anti-IFNg
antibody used, a variety of different cellular constituents were
nonspecifically stained. Consequently, data based on
IFNg-immunohistochemistry have to be interpreted with great caution.

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