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Immunohistochemical localization of TNFalpha and its receptors in the rodent central nervous system.

The protocols for immunohistochemical localization of tumor necrosis factor alpha (TNFalpha), TNFRp55, and TNFRp75 in central nervous system (CNS) slices from laboratory animals are described. The procedure requires intracardiac perfusion of animals followed by fixation and cryostat sectioning in 30-40 microm slices. Free-floating sections are then incubated with the primary antibody followed by incubation with a secondary antibody species-specific for the type of animal in which the primary antibody was made. The secondary antibody is conjugated to biotin for the avidin-biotin complex (ABC) method. The horseradish peroxidase (linked to the ABC complex) allows, in the presence of H2O2, diaminobenzidine oxidation with chromogenic reaction and brown staining. Staining for both TNFalpha and its receptors is normally undetectable in CNS samples from control animals, but it is evident in neuroimmunological or neurodegenerative diseases. The technique is not quantitative, but allows qualitative comparison between samples with light-microscopic cellular resolution.