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Rapid microwave fixation of rat mast cells. I. Localization of granule chymase with an ultrastructural postembedding
Abstract:
We defined the ultrastructural localization of chymase in rat peritoneal mast cells using standard aldehyde fixation and a newly described microwave fixation method (Login GR, Dvorak AM: Microwave energy fixation for electron microscopy. Am J Pathol 120: 230, 1985; Login GR, Stavinoha WB, Dvorak AM: Ultrafast microwave energy fixation for electron microscopy. J Histochem Cytochem 34:381, 1986) and postembedding immunogold labeling. Thin sections were exposed first to goat IgG anti-rat chymase and second to gold-conjugated rabbit Ig directed against goat IgG. By transmission electron microscopy, gold particles were localized to the matrix of cytoplasmic granules. Control sections treated with nonimmune sera did not exhibit labeling of mast cells. Thin sections treated simultaneously with purified rat mast cell chymase and anti-chymase antibody in competition studies, showed a marked reduction in granule staining. These findings demonstrate that a microwave fixation method can be used to rapidly fix cell suspensions for postembedding immunocytochemical studies.
Insights
Microwave fixation rapidly preserves rat peritoneal mast cells for ultrastructural analysis. This method accurately localizes chymase within cytoplasmic granules using immunogold labeling.
Area of Science:
- Cell Biology
- Immunohistochemistry
- Electron Microscopy
Background:
- Mast cells are crucial in allergic and inflammatory responses.
- Chymase is a key enzyme found in mast cell granules.
- Accurate localization of enzymes is vital for understanding cellular function.
Purpose of the Study:
- To determine the ultrastructural localization of chymase in rat peritoneal mast cells.
- To evaluate a novel microwave fixation method for immunoelectron microscopy.
- To compare microwave fixation with standard aldehyde fixation.
Main Methods:
- Rat peritoneal mast cells were fixed using standard aldehyde or microwave methods.
- Postembedding immunogold labeling was performed using anti-rat chymase antibodies.
- Transmission electron microscopy (TEM) was used to visualize gold particle distribution.
Main Results:
- Gold particles, indicating chymase, were localized to the matrix of cytoplasmic granules.
- Microwave fixation allowed for rapid preservation of cell suspensions.
- Control sections and competition studies confirmed the specificity of the antibody labeling.
Conclusions:
- Microwave fixation is a viable and rapid method for preparing cell suspensions for immunoelectron microscopy.
- The study successfully defined the ultrastructural localization of chymase within rat mast cell granules.
- This technique can be applied to other immunocytochemical studies requiring rapid fixation.
