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 IFN gamma immunohis tochemistry on tissue sections with a large panel of anti-IFN gamma antibod ies. Thirteen different commercially available anti-IFN gamma antibodies, i ncluding seven advertised and/or regularly applied for immunohistochemistry /-cytochemistry, were tested using a three-step streptavidin-biotin-peroxid ase technique and a two-step immunofluorescence (FACS) analysis. Immunoenzy me double staining was used to identify the IFN gamma -positive cells. Seri al cryostat sections were used of human reactive hyperplastic tonsils, rheu matoid synovium, and inflammatory abdominal aortic aneurysms, known to poss ess a prominent Th1-type immune response. In vitro phorbol myristate acetat e/ionomycin-stimulated T-cells served as positive control; unstimulated cel ls served as negative control. Cultured T-cells were used adhered to glass slides (immunocytochemistry), in suspension (FACS), or snap-frozen and sect ioned (immunohistochemistry). Immunocytochemistry and FAGS analysis on stim ulated cultured T-cells showed positive staining results with 12 of 13 anti -IFN gamma antibodies. However, immunohistochemistry of sectioned stimulate d T-cells was negative with all. Unstimulated cells were consistently negat ive. lFN gamma 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), extracel lular 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 a ntibodies. IFN gamma -immunohistochemistry appears unreliable because of la ck of specificity to stain T-cells in situ. In fact, depending on the type of anti-IFN gamma antibody used, a variety of different cell constituents w ere nonspecifically stained. Consequently, data based on IFN gamma -immunoh istochemistry must be interpreted with great caution.