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Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry
Published on: June 22, 2017
Optimum immunohistochemical procedures for analysis of macrophages in human and mouse formalin fixed
Takenobu Nakagawa1, Koji Ohnishi1, Yui Kosaki1
1Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University.
Insights
Optimizing immunohistochemistry (IHC) protocols is crucial for studying macrophages in disease. This research provides best practices for antigen retrieval and sample storage, enhancing macrophage analysis in FFPE tissues.
Area of Science:
- Immunology
- Pathology
- Biomedical Research
Background:
- Macrophages play critical roles in various human and mouse diseases.
- Immunohistochemistry (IHC) is essential for evaluating macrophage states in formalin-fixed, paraffin-embedded (FFPE) tissues.
- Standardized IHC protocols are needed for reliable macrophage assessment.
Purpose of the Study:
- To optimize immunohistochemistry (IHC) protocols for macrophage staining.
- To identify optimal antigen retrieval methods for various macrophage markers.
- To determine best practices for long-term storage of tissue sections for IHC analysis.
Main Methods:
- Systematic assessment of different antigen retrieval techniques for macrophage antibodies.
- Evaluation of heat-induced antigen retrieval using ethylenediamine tetraacetic acid (EDTA) buffer at pH 8.0.
- Analysis of immunoreactivity in FFPE tissue sections stored under various conditions, including -80 °C.
Main Results:
- Optimal IHC protocols were established for specific primary antibodies (e.g., mouse anti-Iba1, human anti-CD11b, -CD163, -CD169, -CD204, -CD206).
- Heat treatment with EDTA (pH 8.0) proved effective for multiple macrophage markers.
- Macrophage immunoreactivity in FFPE sections is preserved with long-term storage at -80 °C, but declines over time at other conditions.
Conclusions:
- The study provides optimized IHC protocols for macrophage research.
- Recommended antigen retrieval and storage methods enhance the reliability of macrophage analysis.
- These findings offer valuable tools for researchers studying macrophages in disease contexts.
Abstract:
Macrophages are closely related to various diseases and it is therefore important that the properties of macrophages are adequately evaluated in human diseases and mouse disease models. Immunohistochemistry (IHC) of formalin fixed paraffin-embedded (FFPE) samples is a very useful tool for examination of macrophages; however, an adequate IHC protocol is required for the examination of macrophage states. In this study, we assessed various antigen retrieval methods in order to devise the optimal protocols for staining of macrophages with a range of antibodies. Optimum combinations of primary antibodies and antigen retrieval protocols were determined; for example, heat treatment with ethylenediamine tetraacetic acid solution, pH 8.0, was the best procedure for IHC using mouse anti-Iba1 and human anti-CD11b, -CD163, -CD169, -CD204, and -CD206 antibodies. Moreover, we found that the immunoreactivity of sliced tissue sections decreased gradually over time in long term storage but that this immunoreactivity was preserved in storage at -80 °C in a deep freezer. The optimal IHC protocols and storage procedures that were determined in this study should be a useful tool for macrophage research.
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