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Fixation matters: duration in fixative prior to immunofluorescent analysis directly impacts macrophage visualization
Lizi M Hegarty1, Erin Watson1, Calum C Bain2
1Centre for Regenerative Medicine, Institute for Regeneration and Repair, University of Edinburgh, Edinburgh, UK.
Introduction:
Macrophages are now recognized as key players in a range of tissues and biological processes, responding to injury and infection, and facilitating development and regeneration. As the importance of macrophage crosstalk within these processes has been revealed, so too has the significance of studying the spatial positioning of macrophages within the tissue of interest. As such, immunofluorescent microscopy-based analysis is becoming an increasingly attractive technique for immunology research. While tissue fixation preserves the tissue architecture and immobilizes target antigens, prolonged fixation can negatively impact protein recognition.
Methods:
We compared three different durations of tissue fixation and the expression of key macrophage and structural cell markers in the submandibular gland, pancreas, kidney, and skin by immunofluorescent imaging.
Results:
We report that prolonged exposure to a paraformaldehyde-based fixative profoundly impacts detection of cell surface markers that define macrophage subsets in the mouse submandibular gland, in contrast to epithelial cell markers, which appear more robust. We find that this is not exclusive to the salivary gland, and similar effects are seen in the pancreas and kidney. Importantly, a short duration of fixation allowed the detection of macrophage subsets in both mouse and human tissue without compromising the detection of other markers.
Conclusion:
Adoption of a short fixation approach enables accurate detection of a wide range of cell types in tissues, and facilitates exploration of spatial positioning and cell proximity by immunofluorescent microscopy analysis.
Insights
Short tissue fixation is crucial for accurately detecting macrophage subsets using immunofluorescent microscopy. This method preserves tissue architecture while ensuring reliable detection of cell surface markers in various organs.
Area of Science:
- Immunology
- Cell Biology
- Histology
Background:
- Macrophages are vital immune cells involved in tissue repair, development, and immune responses.
- Understanding macrophage spatial positioning is key to studying their functions in various biological processes.
- Immunofluorescent microscopy is a valuable tool for analyzing macrophage localization within tissues.
Purpose of the Study:
- To investigate the impact of different tissue fixation durations on the detection of macrophage and structural cell markers.
- To determine optimal fixation protocols for immunofluorescent imaging of macrophages in various tissues.
Main Methods:
- Comparison of three tissue fixation durations (short, medium, prolonged).
- Immunofluorescent imaging of macrophage and structural cell markers.
- Analysis in mouse and human tissues, including submandibular gland, pancreas, kidney, and skin.
Main Results:
- Prolonged paraformaldehyde fixation significantly impaired the detection of cell surface markers for macrophage subsets in mouse tissues.
- Epithelial cell markers showed greater robustness to prolonged fixation compared to macrophage markers.
- A short fixation duration enabled accurate detection of macrophage subsets in both mouse and human tissues without compromising other marker detection.
Conclusions:
- Short tissue fixation protocols are essential for accurate immunofluorescent detection of macrophage subsets.
- Optimized fixation enhances the study of macrophage spatial positioning and cell proximity.
- This approach facilitates broader applications of immunofluorescent microscopy in immunology research.
