Immunohistochemical detection of interferon-gamma: fake or fact?
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 IFNgamma immunohistochemistry on tissue sections with a large panel of anti-IFNgamma antibodies. Thirteen different commercially available anti-IFNgamma antibodies, including seven advertised and/or regularly applied for immunohistochemistry/-cytochemistry, were tested using a three-step streptavidin-biotin-peroxidase technique and a two-step immunofluorescence (FACS) analysis. Immunoenzyme double staining was used to identify the IFNgamma-positive cells. Serial cryostat sections were used of human reactive hyperplastic tonsils, rheumatoid synovium, and inflammatory abdominal aortic aneurysms, known to possess a prominent Th1-type immune response. 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 snap-frozen and sectioned (immunohistochemistry). Immunocytochemistry and FACS analysis on stimulated cultured T-cells showed positive staining results with 12 of 13 anti-IFNgamma antibodies. However, immunohistochemistry of sectioned stimulated T-cells was negative with all. Unstimulated cells were consistently negative. IFNgamma 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. IFNgamma-immunohistochemistry appears unreliable because of lack of specificity to stain T-cells in situ. In fact, depending on the type of anti-IFNgamma antibody used, a variety of different cell constituents were nonspecifically stained. Consequently, data based on IFNgamma-immunohistochemistry must be interpreted with great caution.
van der Loos
C M
CM
Academic Medical Center, Department of Cardiovascular Pathology, Amsterdam, The Netherlands. c.m.vanderloos@amc.uva.nl
Houtkamp
M A
MA
de Boer
O J
OJ
Teeling
P
P
van der Wal
A C
AC
Becker
A E
AE
D000368:Aged; D000918:Antibody Specificity; D001014:Aortic Aneurysm / Q000276:immunology / Q000473:pathology; D001172:Arthritis, Rheumatoid / Q000276:immunology / Q000473:pathology; D002648:Child; D002675:Child, Preschool; D005434:Flow Cytometry; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods; D007150:Immunohistochemistry / Q000379:methods*; D007202:Indicators and Reagents / Q000592:standards; D007371:Interferon-gamma / Q000276:immunology* / Q000302:isolation & purification*; D008875:Middle Aged; D014066:Palatine Tonsil / Q000276:immunology / Q000473:pathology; D015203:Reproducibility of Results; D018417:Th1 Cells
D007202:Indicators and Reagents; D007371:Interferon-gamma