Tumor Necrosis Factor- Quantification and Expression by In Situ Hybridization

The pleiotropic cytokine, tumor necrosis factor-α (TNF-α), has been shown to affect not only pathological and inflammatory conditions, but also plays a seminal role in the maintenance of physiological homeostasis. Due to these diverse activities, TNF-α is widely studied using various assay methods. The WEHI 164 clone 13 (1 ) and WEHI-13VAR (2 ) bioassays are the method of choice for detection of biologically active TNF in biological fluids and tissue homogenates (3 –4 ). However, data generated from these assays are limited, providing only bioactive levels of this cytokine in a whole tissue homogenate or in serum/plasma. In order to determine the cellular localization for production of this cytokine, the techniques of immunohistochemistry and in situ hybridization are invaluable. These methods allow for the successful identification of the cell types involved in the regulation and production of this cytokine. We describe modification of both methodologies that allows for detection of TNF-α protein as well as the messenger RNA (5 –7 ) for this protein in rat brain tissue preparations.