Immunohistochemical Localization of TNFα and Its Receptors in the Rodent Central Nervous System

The protocols for immunohistochemical localization of tumor necrosis factor α (TNFα), 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 μm 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 H2 O2 , diaminobenzidine oxidation with chromogenic reaction and brown staining. Staining for both TNFα 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.