Encyclopedia of Experiments: Immunology
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Transcript
Begin with a glass slide carrying a deparaffinized and rehydrated infected urinary bladder biopsy tissue section containing uropathogenic bacteria.
Introduce fluorescently-labeled universal probes — a complementary single-stranded oligonucleotide specific to the conserved region of bacterial 16s ribosomal RNA, or 16s-rRNA.
Incubate the slide in the dark at an elevated temperature for optimum hybridization.
During incubation, the probes exclusively hybridize to the conserved region on bacterial 16s-rRNA, imparting green fluorescence to the bacteria.
Post-hybridization, wash with a pre-warmed buffer to remove the unbound probes.
Stain the tissue section with Hoechst dye and fluorophore-labeled cellular protein markers.
Hoechst molecules bind to the DNA, staining the nuclei blue, while fluorophore-labeled markers interact with specific cell components such as mucin and actin filaments, imparting red fluorescence in the biopsy section.
Wash the slide and add mounting media to the tissue section.
Under a confocal microscope, green fluorescence within the red fluorescence-carrying bladder biopsy section confirms the tissue-associated bacteria.
