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A solvent-based fixative for electron microscopy to improve retention and visualization of the intestinal mucus
P Allan-Wojtas1, E R Farnworth, H W Modler
1Centre for Food and Animal Research, Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada.
Abstract:
Samples of pig small intestine, cecum, and large intestine were prepared for scanning electron microscopy (SEM), concentrating on mucus blanket retention and visualization. Samples were fixed using three aqueous-based fixatives which included a standard glutaraldehyde fixative alone as the control and the standard fixative formulation with either ruthenium red or alcian blue added and using one solvent-based fixative, osmium tetroxide dissolved in FC-72 (a degreasing fluorocarbon solvent produced by 3M Canada, Inc.), which had been successfully used by Sims et al. [(1991) Biotech. Histochem., 66:173-180] to preserve tracheal mucus of nonhuman mammals. Pig intestine samples prepared using the solvent-based fixative retained a contiguous mucus blanket, while the aqueous-based treatments retained only patchy or fibrous remnants to a degree depending on fixative composition and intestinal site. We conclude that preparation of the pig intestinal mucus layer using the solvent-based fixative suggested by Sims et al. (1991) preserves the mucus blanket in its entirety and gives superior results to aqueous-based fixatives containing the standard additives ruthenium red and alcian blue. We recommend that this anhydrous fixation, which requires only a slight modification from standard conditions, be adopted when mucus layer retention and visualization is important, as in the field of probiotics. Overcoming this major technical obstacle will now allow electron microscopy (EM) to once again provide new in situ information in this reemerging field.
Insights
A new solvent-based fixative effectively preserves the entire mucus layer in pig intestines for scanning electron microscopy (SEM). This method significantly improves visualization of the mucus blanket compared to traditional aqueous fixatives, aiding research in fields like probiotics.
Area of Science:
- Gastroenterology
- Microscopy techniques
- Histology
Background:
- Preserving the intestinal mucus layer is crucial for understanding gut health and microbial interactions.
- Traditional aqueous-based fixatives often fail to retain the integrity of the mucus blanket for electron microscopy.
- Previous methods have limitations in visualizing the complete mucus layer in the pig intestine.
Purpose of the Study:
- To evaluate and compare the efficacy of different fixatives for preserving the pig intestinal mucus layer for scanning electron microscopy (SEM).
- To identify an optimal fixation method for visualizing the entire mucus blanket in situ.
- To overcome technical challenges in studying the intestinal mucus layer for fields like probiotics.
Main Methods:
- Samples of pig small intestine, cecum, and large intestine were prepared using four fixatives: glutaraldehyde (control), glutaraldehyde with ruthenium red, glutaraldehyde with alcian blue, and osmium tetroxide in FC-72 (solvent-based).
- Scanning electron microscopy (SEM) was used to examine mucus blanket retention and visualization.
- Comparison of mucus layer integrity across different intestinal sites and fixation methods.
Main Results:
- The solvent-based fixative (osmium tetroxide in FC-72) successfully retained a contiguous mucus blanket across all tested pig intestinal sections.
- Aqueous-based fixatives resulted in patchy or fibrous mucus remnants, with variable retention depending on composition and intestinal site.
- The solvent-based method provided superior preservation of the mucus layer compared to aqueous fixatives with ruthenium red or alcian blue.
Conclusions:
- Anhydrous fixation using osmium tetroxide in FC-72 is highly effective for preserving the entire pig intestinal mucus layer for SEM.
- This method offers significant advantages over standard aqueous-based fixatives for mucus visualization.
- The improved technique facilitates in situ studies of the mucus layer, particularly relevant for probiotic research and other fields utilizing electron microscopy (EM).