Fixation matters: duration in fixative prior to immunofluorescent analysis directly impacts macrophage visualisation in epithelial tissues

Abstract Introduction Macrophages are now recognised 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 immobilises target antigens, prolonged fixation can negatively impact protein recognition. Methods We compared three different durations of tissue fixation and compared 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.