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Combined immunohistochemical and immunofluorescence method to determine the phenotype of proliferating cell
1Nuffield Department of Pathology and Bacteriology, John Radcliffe Hospital, University of Oxford.
Insights
A novel double immunostaining technique allows simultaneous identification of cell markers and proliferation in human tissues. This method effectively visualizes cell phenotypes in tonsil and atherosclerotic tissues.
Area of Science:
- Immunohistochemistry
- Cell Biology
- Pathology
Background:
- Characterizing proliferating cell populations is crucial for understanding tissue dynamics in health and disease.
- Existing immunostaining methods can be time-consuming and may not allow for simultaneous detection of multiple markers.
Purpose of the Study:
- To develop and validate a novel double immunostaining technique for determining the phenotype of proliferating cell populations.
- To assess the utility of this technique in human tonsil and atherosclerotic aortic aneurysm tissues.
Main Methods:
- A double immunostaining method was developed combining autofluorescent alkaline phosphatase substrate (naphthol/Fast Red) with immunofluorescence (fluorescein).
- Human tonsil and atherosclerotic aortic aneurysm tissues were analyzed using monoclonal antibodies against Ki-67, CD4, CD8, CD19, CD22, HLA-DR alpha, CD68, and CD31.
Main Results:
- The technique enabled simultaneous colocalization of different markers on the same cell.
- Proliferation-associated Ki-67 positive cells and HLA-DR positive cells were successfully identified in human tonsil and atherosclerotic tissues.
- The method demonstrated effectiveness in identifying cell phenotypes within complex tissue environments.
Conclusions:
- This double immunostaining technique is simple, efficient, and allows for the simultaneous identification of multiple cell markers and proliferation status.
- The stable Fast Red reaction product and compatibility with nuclear, cytoplasmic, and surface antigens make it a versatile tool.
- The technique offers a valuable approach for studying cell phenotypes in various human tissues, including those affected by atherosclerosis.
Aims:
To determine the phenotype of proliferating cell populations.
Methods:
The double immunostaining technique combines the autofluorescent properties of alkaline phosphatase substrate naphthol/Fast Red with immunofluorescence using fluorescein. Fresh human tonsil and fresh atherosclerotic aortic aneurysm wall tissue were studied using a panel of monoclonal antibodies including Ki-67, CD4, CD8, CD19, CD22, HLA-DR alpha, CD68 and CD31.
Results:
This double immunostaining method permitted simultaneous colocalisation of different markers on the same cell and could be used to identify HLA-DR positive cells as well as proliferation associated Ki-67 positive cells in human tonsil tissue and in chronic periaortitis associated with advanced atherosclerosis.
Conclusion:
This technique is simple and the results may be viewed using a single fluorescence filter. The Fast Red reaction product is stable and does not fade under storage. The staining works particularly well with markers for nuclear antigens in combination with markers for cytoplasmic or surface antigens.
