Abstract
Preservation of three-dimensional structure in the gut is necessary in order to analyze the spatial organization of the gut microbiota and gut luminal contents. In this study, we evaluated preparation methods for mouse gut with the goal of preserving micron-scale spatial structure while performing fluorescence imaging assays. Our evaluation of embedding methods showed that commonly used media such as Tissue-Tek Optimal Cutting Temperature (OCT) compound, paraffin, and polyester waxes resulted in redistribution of luminal contents. By contrast, a hydrophilic methacrylate resin, Technovit H8100, preserved three-dimensional organization. Our mouse intestinal preparation protocol optimized using the Technovit H8100 embedding method was compatible with microbial fluorescence in situ hybridization (FISH) and other labeling techniques, including immunostaining and staining with both wheat germ agglutinin (WGA) and 4', 6-diamidino-2-phenylindole (DAPI). Mucus could be visualized whether the sample was fixed with paraformaldehyde (PFA) or with Carnoy’s fixative. The protocol optimized in this study enabled simultaneous visualization of micron-scale spatial patterns formed by microbial cells in the mouse intestines along with biogeographical landmarks such as host-derived mucus and food particles.
Highlights
Preservation of spatial structure in intestinal samples is crucial for investigating spatial organization of the gut microbiota relative to mucins, host tissue, and food particles
A major finding of this study is that embedment methods differ in how well they preserve the three-dimensional organization of the gut luminal contents including microbes, food particles and mucus
Our results indicate that the protocols involving commonly used embedment media, Tissue-Tek Optimal Cutting Temperature (OCT) compound, paraffin or polyester waxes allowed the luminal contents to become lost or redistributed, for reasons that seem evident
Summary
Preservation of spatial structure in intestinal samples is crucial for investigating spatial organization of the gut microbiota relative to mucins, host tissue, and food particles. Selecting a protocol for intestinal sample preparation is made difficult by conflicting recommendations in the literature. Several authors report that fixation in aqueous fixatives such as formaldehyde results in collapse or loss of the mucus layer and suggest that fixation in the non-aqueous Carnoy or methacarn solution is essential for mucus preservation [1,2,3]. A widely used protocol is to process gut samples by Carnoy fixation followed by paraffin embedding and sectioning. Report that the use of organic solvents during the paraffin clearing step results in extraction of portions of the mucus layer and instead recommend.
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