We present here sensitive, simple and robust methods for detection of tumor necrosis factor (TNF) mRNA and TNF in histological sections and homogenates of brain tissue from mice subjected to focal cerebral ischemia or hippocampal axonal lesioning. Both types of lesions are characterized by induction of TNF synthesis in resident microglial cells, which in the ischemic lesions are supplemented by TNF synthesizing, blood-borne macrophages. In situ hybridization for TNF mRNA is performed using alkaline phosphatase-labelled oligodeoxynucleotide probes. These probes show excellent rendition of individual cells, and can successfully be combined with immunohistochemical procedures. We also describe a sensitive immunohistochemical method for detection of TNF, which can be combined with visualization of an additional antigen. The specificity of the histological procedures are confirmed by RT-PCR and Western blot analysis on homogenates prepared from microdissected brain regions. Advantages and disadvantages of the methods are discussed with emphasis on the specificity and sensitivity of the histological procedures. Our strategy for detection of TNF mRNA and protein provides a solid basis for clarifying the cellular synthesis, regulation and function of TNF in the normal, injured or diseased CNS. Furthermore, the methodology can readily be applied in studies of other cytokines and growth factors in the CNS.
Brain Research Protocols, 2001, Vol 7, Issue 2, p. 175-91