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Immune complex receptors on cell surface. I. Ultrastructural demonstration of macrophages
Abstract:
A method is described for ultrastructural localization of immune complex receptors on the surface of viable peritoneal exudate cells. The technique entails incubation with a soluble complex of horseradish peroxidase (HRP) and specific antibody to HRP at 4 degrees C followed by exposure to diaminobenzidine and processing for electron microscopy. The bound immune complexes were evident as focal deposits of HRP reaction product, adhering closely to the external surface of macrophages with an uninterrupted periodicity varying between 30 and 120 nm. Following incubation with an insoluble immune complex containing a higher proportion of antibody, receptor sites stained frequently, but large aggregates adhered to the cells. Rinsing cells after staining with soluble complexes partially displaced the bound immune complexes. Fixation prior to exposure to immune complexes largely eliminated the binding capacity of the immune complex receptors.
Insights
This study presents a new method for visualizing immune complex receptors on living cells using horseradish peroxidase (HRP) and electron microscopy. The technique successfully localized these receptors on macrophages, aiding in understanding cellular immune responses.
Area of Science:
- Immunology
- Cell Biology
- Microscopy
Background:
- Immune complex receptors play a crucial role in cellular immunity.
- Accurate localization of these receptors is vital for understanding their function.
- Existing methods may have limitations in preserving cell viability and receptor integrity.
Purpose of the Study:
- To develop and validate a novel method for ultrastructural localization of immune complex receptors on viable peritoneal exudate cells.
- To assess the binding characteristics and distribution of immune complex receptors on macrophages.
Main Methods:
- Incubation of viable peritoneal exudate cells with a soluble complex of horseradish peroxidase (HRP) and anti-HRP antibody at 4°C.
- Staining with diaminobenzidine (DAB) to visualize HRP activity.
- Processing for electron microscopy to observe ultrastructural localization.
Main Results:
- Bound immune complexes were observed as focal deposits of HRP reaction product on the macrophage surface.
- A periodicity of 30-120 nm was noted between bound immune complexes.
- Fixation prior to immune complex exposure significantly reduced receptor binding capacity, while rinsing after soluble complex staining partially displaced bound complexes.
Conclusions:
- The described method allows for effective ultrastructural localization of immune complex receptors on viable cells.
- The technique highlights the dynamic nature of immune complex binding and receptor accessibility.
- Preserving cell viability during the procedure is critical for accurate receptor localization studies.