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Updated: Mar 22, 2026
Identification of Neutrophil Extracellular Traps in Paraffin-Embedded Feline Arterial Thrombi using Immunofluorescence Microscopy
Published on: March 29, 2020
Development of a fluorescence in situ hybridization protocol for use with formalin-fixed paraffin-embedded samples of
Matthew J Rolph1, Pompei Bolfa2, Sarah M Cavanaugh1
1Departments of Clinical Sciences, Ross University School of Veterinary Medicine, Basseterre, St Kitts, West Indies.
Abstract:
Fluorescence in situ hybridization (FISH) can be used to detect intracellular bacteria in formalin-fixed paraffin-embedded (FFPE) tissues. However, this technique has rarely been applied to samples of feline heart, liver, or kidney to investigate the relationship between intracellular bacteria and chronic inflammation within these organs. Our objective was to develop a robust FISH protocol to detect intracellular bacteria within medium-density FFPE feline tissue samples. Cy3-labeled eubacterial and non-eubacterial probes were applied to samples of monkey intestine, feline intestine, and feline liver, in which bacteria had been visualized on routine histopathology. Although initial tests failed, the addition of pepsin pre-digestion to the test protocol and the adjustment of stringency ensured consistent results. All positive results were confirmed using Gram staining and non-eubacterial probes applied to sequential samples of the 3 tissues. Our FISH protocol using Cy3-labeled eubacterial probes reliably and consistently detected bacteria within FFPE samples of the 3 tissues. The addition of 20-min pepsin pre-digestion and the adjustment of the formamide concentration within the hybridization buffer to 40% were pivotal to the successful use of the protocol. Species-specific probes, along with PCR, culture, and special staining, could be considered to increase sensitivity and specificity of FISH when used for the detection of organisms within tissues.
Insights
This study developed a reliable fluorescence in situ hybridization (FISH) protocol to detect intracellular bacteria in feline FFPE tissues. The optimized FISH method enhances bacterial detection in feline organs, aiding research into chronic inflammation.
Area of Science:
- Veterinary Pathology
- Microbiology
- Molecular Biology
Background:
- Intracellular bacteria in feline organs are linked to chronic inflammation.
- Fluorescence in situ hybridization (FISH) is a potential tool for detecting these bacteria.
- Limited studies have applied FISH to feline heart, liver, or kidney tissues.
Purpose of the Study:
- To develop and optimize a robust FISH protocol for detecting intracellular bacteria in formalin-fixed paraffin-embedded (FFPE) feline tissues.
- To investigate the presence of bacteria in feline heart, liver, and kidney samples.
Main Methods:
- Development of a FISH protocol using Cy3-labeled eubacterial probes.
- Application of the protocol to FFPE feline liver, intestine, and monkey intestine samples.
- Optimization involved pepsin pre-digestion and adjusted hybridization stringency (40% formamide).
- Results were confirmed using Gram staining and non-eubacterial probes.
Main Results:
- The optimized FISH protocol reliably and consistently detected intracellular bacteria in FFPE feline tissues.
- Pepsin pre-digestion (20 minutes) and 40% formamide concentration were critical for success.
- Successful detection was confirmed across feline and monkey intestinal and feline liver tissues.
Conclusions:
- A robust FISH protocol was successfully established for detecting intracellular bacteria in FFPE feline tissues.
- This method can aid in understanding the role of bacteria in feline chronic inflammatory diseases.
- Further enhancements using species-specific probes and complementary techniques can improve sensitivity and specificity.
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