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
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Updated: Aug 14, 2026
Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays
Published on: July 4, 2018
Abstract:
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-alpha 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-alpha immunoreactivity. We also calculated the density of TNF-alpha labeling/mu 2 in both dense (unaltered) and altered granules in specimens. TNF-alpha 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-alpha label. These findings show that changes in the immunolocalization and/or density of TNF-alpha immunoreactivity occur very rapidly upon stimulation of rat peritoneal mast cells with compound 48/80.
Insights
Stimulating rat peritoneal mast cells rapidly alters cytoplasmic granules and decreases tumor necrosis factor-alpha (TNF-alpha) immunoreactivity within them. These rapid changes highlight early cellular responses to secretagogues.
Area of Science:
- Cell Biology
- Immunology
- Histology
Background:
- Mast cells are key immune cells involved in inflammatory responses.
- Tumor necrosis factor-alpha (TNF-alpha) is a critical inflammatory mediator released by mast cells.
- Understanding mast cell degranulation dynamics is crucial for inflammatory disease research.
Purpose of the Study:
- To investigate rapid morphological changes in rat peritoneal mast cells after stimulation.
- To examine the early changes in TNF-alpha immunoreactivity within mast cell cytoplasmic granules.
- To assess the impact of microwave-assisted fixation on preserving ultrastructural details and antigenicity.
Main Methods:
- Utilized fast (seconds) and ultrafast (milliseconds) microwave energy-assisted chemical fixation.
- Employed postembedding immunogold staining for TNF-alpha detection.
- Conducted morphometric analysis to quantify granule changes and TNF-alpha labeling density.
Main Results:
- Compound 48/80 stimulation led to increased, swollen cytoplasmic granules with decreased electron density.
- A significant reduction in TNF-alpha immunoreactivity was observed in these altered granules.
- TNF-alpha was present in dense granules but its labeling density decreased in altered granules.
Conclusions:
- Mast cell stimulation by compound 48/80 induces rapid, detectable morphological changes in granules.
- TNF-alpha immunolocalization and/or density changes occur very quickly upon mast cell activation.
- Microwave fixation effectively preserves cellular structures and TNF-alpha antigenicity for rapid analysis.
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