Source: Neugent, M. L. et al., Detection of Tissue-resident Bacteria in Bladder Biopsies by 16S rRNA Fluorescence In Situ Hybridization. J. Vis. Exp. (2019)
This video demonstrates the visualization of bacteria in deparaffinized bladder tissue from a urinary tract-infected patient. Fluorescently-labeled probe hybridized with bacterial 16s ribosomal RNAs reveals the tissue-associated bacteria and their distribution, providing valuable spatial insights into their density within the sample.
All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.
1. Tissue Preparation for Fixation and Paraffin Embedding
NOTE: Biopsies were taken from consenting women undergoing cystoscopy with electro-fulguration of trigonitis for the advanced management of recurrent urinary tract infection (rUTI). rUTI is defined as 3 UTIs in 12 months. Biopsy collection was performed in the operating room while the patient was under anesthesia after obtaining informed patient consent. All samples were coded and de-identified before experimentation.
2. Fluorescence In Situ Hybridization with Universal 16S rRNA Probes
NOTE: Two slides per biopsy are required. One slide is needed for the universal 16S rRNA probe and one slide for a control probe with a scrambled sequence. This is important for distinguishing the true signal from the background signal during microscopy since the bladder epithelium is auto-fluorescent in multiple channels. In addition to the scramble probe, blocking with 0.1% Sudan Black B prior to mounting may reduce background autofluorescence inherent in the tissue.
3. Visualization of 16S rRNA FISH by Confocal Microscopy
NOTE: For this protocol, best results are achieved with a laser-scanning confocal microscope with 63x and 100x objectives. Proper filter sets for visualization of Hoechst, Alexa-488, and Alexa-555 fluorescence are required. However, standard fluorescent microscopy can be used if a confocal microscope is unavailable. This protocol is for a laser scanning confocal microscope.
The authors have nothing to disclose.
Alexa-555 Phalloidin | Invitrogen | A34055 | Staining Actin |
Alexa-555 Wheat Germ Agglutinin (WGA) | Invitrogen | W32464 | Staining Mucin |
Bottle top filters | Fisher Scientific | 09-741-07 | Sterilization |
Coplin Jar | Simport | M900-12W | Deparaffinization/washing |
Coverslips | Fisher Scientific | 12-548-5M | Microscopy |
Ethanol | Fisher Scientific | 04-355-224 | Rehydration |
Ethylenediaminetetraacetic Acid, Disodium Salt Dihydrate | Fisher Scientific | S311-500 | TE |
Frosted Slides | Thermo Fisher Scientific | 12-550-343 | |
Hoechst 33342, Trihydrochloride, trihydrate | Invitrogen | H21492 | Staining Nucleus |
Hydrophobic marker | Vector Laboratories | H-4000 | Hydrophobic barrier |
Kimwipes | Fisher Scientific | 06-666-11 | |
Oil Immersol 518 F | Fisher Scientific | 12-624-66A | Microscopy |
Paraformaldehyde (16%) | Thermo Scientific | TJ274997 | Fixation |
ProLong Gold antifade reagent | Invitrogen | P36934 | Mounting medium |
Sodium Chloride | Fisher Scientific | BP358-10 | Hybridization buffer/PBS |
Sodium Dodecyl Sulphate | Fisher Scientific | BP166-500 | Hybridization buffer |
Sodium Phosphate Dibasic Hepahydrate | Fisher Scientific | S373-500 | PBS |
Sodium Phosphate Monobasic Monohydrate | Fisher Scientific | S369-500 | PBS |
Syringe | VWR | 75486-756 | Sterilization |
Tris-HCl | Fisher Scientific | BP152-5 | TE/Hybridization buffer |
Xylene | Fisher Chemical | X3P-1GAL | Deparaffinization |
0.22 micron syringe filter | Fisher Scientific | 09-754-29 | Sterilization |