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Published on: December 15, 2017
Non-Specific Binding, a Limitation of the Immunofluorescence Method to Study Macrophages In Situ
Emma Sicherre1, Anne-Laure Favier1, Diane Riccobono2
1Imagery Unit, Department of Platforms and Technology Research, French Armed Forces Biomedical Research Institute, 91223 Brétigny-sur-Orge, France.
Insights
Characterizing in vivo macrophages is crucial for tissue regeneration. This study found that CD206 antibody variations can yield false macrophage detection, highlighting in situ hybridization with hybridization-chain-reaction detection (HCR) as a reliable alternative.
Area of Science:
- Immunology
- Regenerative Medicine
- Cell Biology
Background:
- In vivo macrophage characterization is essential for understanding tissue regenerative mechanisms.
- The immuno-fluorescence method using pan-markers like CD206 protein is a common approach.
- Accurate macrophage identification is critical for researchers in regenerative medicine.
Purpose of the Study:
- To investigate CD206 protein expression in an irradiated-muscle pig model.
- To evaluate the reliability of different antibodies for macrophage immunodetection.
- To identify alternative methods for accurate in situ macrophage detection.
Main Methods:
- Utilized immuno-fluorescence with three distinct CD206 antibodies in an irradiated-muscle pig model.
- Employed in situ hybridization coupled with hybridization-chain-reaction (HCR) detection.
- Compared the expression patterns and reliability of different detection methods.
Main Results:
- Observed differing CD206 expression patterns based on antibody origin during immunodetection.
- Identified potential for false-positive or false-negative results with certain CD206 antibodies.
- Demonstrated that in situ hybridization with HCR provides accurate in situ macrophage detection.
Conclusions:
- Antibody variability can significantly impact the accuracy of macrophage characterization using CD206.
- False results in macrophage immunodetection are a potential pitfall for researchers.
- In situ hybridization with HCR offers a robust and reliable alternative for in situ macrophage identification.
Abstract:
Advances in understanding tissue regenerative mechanisms require the characterization of in vivo macrophages as those play a fundamental role in this process. This characterization can be approached using the immuno-fluorescence method with widely studied and used pan-markers such as CD206 protein. This work investigated CD206 expression in an irradiated-muscle pig model using three different antibodies. Surprisingly, the expression pattern during immunodetection differed depending on the antibody origin and could give some false results. False results are rarely described in the literature, but this information is essential for scientists who need to characterize macrophages. In this context, we showed that in situ hybridization coupled with hybridization-chain-reaction detection (HCR) is an excellent alternative method to detect macrophages in situ.
