USE OF A RAPID MICROWAVE FIXATION TECHNIQUE FOR IMMUNOCYTOCHEMICAL DEMONSTRATION OF TUMOR NECROSIS FACTOR, INTERLEUKIN-1α, AND INTERLUEKIN-1β IN ACTIVATED HUMAN PERIPHERAL MONONUCLEAR CELLS. (<Symposium>Microwave Fixation in Histochemistry)

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In the present paper, we demonstrated the presence of various cytokines in activated mononuclear cells immunocytochemically by using a rapid microwave (MW) fixation technique. Cytospin preparations of human peripheral blood mononuclear cells activated by a biological response modifier, OK-432, in vitro, were quickly fixed by MW irradiation in a diluted aldehyde solution consisting of 0.05% glutaraldehyde, 2% formaldehyde, 0.025% calcium chloride and 0.1 M sodium cacodylate in phosphate-buffered saline (PBS) (pH 7.4). In the stimulated cells, tumor necrosis factor-α (TNF), interleukin-1α (IL-1α), and interleukin-1β(IL-179) were clearly demonstrated immunocytochemically by using the specific antibodies for each cytokine. Immunoreactivity for TNF appeared in the cells after 1.5 hr of stimulation, and diffuse cytoplasmic stain was achieved after 24 hr of stimulation. IL-1α appeared in both cytoplasms and nuclei of the stimulated cells after 3 hr. The distribution of cytoplasmic IL-1α suggested the association with the cytoplasmic membrane. With IL-1β, a strong diffuse stain was obtained after 6 hr and indicated that the IL-1β was concentrated in cytoplasm. Rapid MW fixation makes it possible to fix metabolically labile cytokines which have been difficult to localize in routinely fixed specimens. The method is expected to be a valuable tool in the study of cell biology.
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