Tumor Necrosis Factor Alpha Immunoreactivity of Rat Peritoneal Mast Cell Granules Decreases during Early Secretion Induced by Compound 48/80: An Ultrastructural Immunogold Morphometric Analysis

We used fast (seconds) and ultrafast (milliseconds) microwave energy-assisted chemical fixation protocols, postembedding immunogold staining, and a morphometric analysis to investigate the early morphological changes and the TNF-α immunoreactivity in the cytoplasmic granules of rat peritoneal mast cells that had been stimulated to secrete by exposure to compound 48/80. Exposure to compound 48/80 induced the development of increased numbers of cytoplasmic granules that exhibited decreased electron density; these granules often also appeared swollen. These granule alterations were accompanied by a significantly decreased proportion of granules that were positive for TNF-α immunoreactivity We also calculated the density of TNF-α labeling/μ2 in both dense (unaltered) and altered granules in specimens. TNF-α immunoreactivity was present in dense granules (regardless of whether or not the specimens had been stimulated with compound 48/80) and in cells that were fixed with either fast or ultrafast microwave energy. However, altered granules exhibited a decreased density of TNF-α label. These findings show that changes in the immunolocalization and/or density of TNF-α immunoreactivity occur very rapidly upon stimulation of rat peritoneal mast cells with compound 48/80.