Development of a fluorescence in situ hybridization protocol for use with formalin-fixed paraffin-embedded samples of feline heart,liver,and kidney

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.