We would like to thank Kim Orth and Marcela de Souza Santos for protocol advice and Amanda Arute for technical support. This work was partially supported by the Cecil H. and Ida Green Chair in Systems Biology Science held by K.P.

The study protocol was approved by and followed the guidelines of the Institutional Biosafety and Chemical Safety Committees of UT Dallas and UT Southwestern Medical Center. The use of biopsies from human subjects in this protocol was approved by and followed the guidelines of the Institutional Review Boards of the UT Dallas and the UT Southwestern Medical Center. All individuals involved with biopsy collection and processing have current human subject protection (HSP) and HIPPA training.
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 a 12-month period. Biopsy collection was performed in the operating room while the patient was under anesthesia after obtaining informed patient consent per UTSW IRB protocol STU 082010-016. All samples were coded and de-identified before experimentation.
<ol>
	<li>Obtain a cold-cup (~1 mm3) biopsy from the bladder trigone with urologic forceps via flexible cystoscope and place immediately into a sterile 2 mL cryovial containing 1.5 mL of 4% v/v paraformaldehyde (PFA) prepared in 1x sterile phosphate buffered saline (PBS). 
	NOTE: 4% PFA in 1x PBS can be made in advance and stored at -20 &#176;C until needed.</li>
	<li>Fix biopsy for 6 h at room temperature or for 16-24 h at 4 &#176;C. 
	NOTE: Fixation duration should be calculated based on the size of the tissue sample and over-fixation should be avoided. It is not advised to use glutaraldehyde as a fixative as it introduces autofluorescence.</li>
	<li>In a sterilized hood or biosafety cabinet, remove fixative by pipetting and replace with 1.5 mL of sterile 1x PBS. Keep the samples at 4 &#176;C overnight or immediately perform tissue processing and paraffin embedding19.</li>
	<li>Use a sterilized microtome to section tissue blocks at 5 &#956;m thickness and adhere paraffin tissue sections to charged glass microscope slides. A minimum of two slides per biopsy will be required for the 16S rRNA FISH protocol. 
	NOTE: The biopsy tissue should be cross sectionally arranged relative to the cutting plane in order to ensure visualization of urothelial layers in all sections. It should also be noted that section thickness should be optimized for bacterial community detection. Thinner sections or sampling of multiple serial sections may be required in the case of infections where tissue-resident bacteria are extremely scarce (e.g., &#60;1 tissue-resident bacterium per 1,000 mammalian cells).</li>
</ol>
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 true signal from 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 tissue20.
<ol>
	<li>Preparation of reagents and fume hood
	<ol>
		<li>Clean an empty fume hood (or appropriately fitted biosafety cabinet) with 70% ethanol.</li>
		<li>Prepare the hybridization buffer comprised of 0.9 M sodium chloride (NaCl), 20 mM Tris-HCl (pH 7.2), 0.1% sodium dodecyl sulfate (SDS) in 10 mL of sterile-filtered water. 
		NOTE: Sterile-filtered water may be prepared by passing autoclaved distilled water through a 0.22 &#956;M filter. The hybridization buffer can be stored at room temperature, but the SDS may precipitate from solution. If SDS precipitates, warm the solution in a 50 &#176;C water bath prior to use.</li>
		<li>Prepare at least 100 mL each of 95% and 90% ethanol in sterile-filtered water in washed, autoclaved bottles.</li>
		<li>Dissolve fluorescently labeled lyophilized probes in 10 mM Tris-HCl (pH 8.0) and 1 mM ethylenediaminetetraacetic acid (EDTA) buffer (TE) prepared in filter-sterilized nuclease free water to a final concentration of 100 &#956;M. Prepare a dilution of 1 &#956;M in TE buffer for use in this protocol. Store both 100 &#956;M concentrated stock and 1 &#956;M stock reconstituted probes at -20 &#176;C protected from light. 
		NOTE: Do not dissolve fluorescently labeled probes in water. Buffering is required to prevent hydrolysis of the NHS ester bond conjugating the fluorophore to the nucleotide probe.</li>
		<li>Clean five Coplin jars with 70% ethanol, allow to dry, label as follows: xylenes I, xylenes II, 95% EtOH, 90% EtOH, ddH2O, and fill with 100 mL of the appropriate solution. This will help avoid confusion in later steps.</li>
	</ol>
	</li>
	<li>Tissue de-paraffinization and rehydration
	<ol>
		<li>In the hood, place two slides per biopsy into a vertical slide rack.</li>
		<li>Place a vertical slide rack into the xylenes I Coplin jar (containing 100 mL of xylenes) for 10 min.</li>
		<li>Remove the slide rack from xylenes I and blot the bottom on a paper towel to remove excess xylenes. Place into the xylenes II Coplin jar for 10 min. 
		NOTE: Never work with xylenes outside of a certified fume hood.</li>
		<li>Rehydrate deparaffinized tissue sections in successive ethanol washes (95% and 90%) for 10 min each in the respectively labeled Coplin jars. 
		NOTE: During this stage, warm Hybridization Buffer to 50-56 &#176;C in a water bath</li>
		<li>Remove the slide rack from the 90% ethanol wash, blot the bottom on paper towels to remove excess ethanol, and place in the ddH2O Coplin jar containing 100 mL of filter-sterilized ddH2O for 10 min.</li>
		<li>While waiting for the ddH2O wash, dilute the probes to 10 nM in hybridization buffer to create the staining solution. Prepare 150 &#956;L of staining solution per slide. 
		NOTE: Protect staining solution from light by wrapping the tubes in aluminum foil and storing in a drawer. When working with fluorescently labeled probes on the benchtop, consider turning off overhead lights when able. Probes diluted in hybridization buffer should not be re-used.</li>
	</ol>
	</li>
	<li>Hybridization and counter-staining
	<ol>
		<li>Prepare a humidifying chamber for each probe by placing soaked, crumpled Kimwipe and sterile water to the reservoir of a P1000 tip box. Place the tip holder cartridge on-top - this is where the slides will sit. 
		NOTE: It is important to use a humidifying chamber to prevent the biopsy sections from drying out during hybridization. It should be noted that commercially available humidifier chambers are designed to maintain a stable, humid atmosphere. However, the technique detailed here sufficiently controls humidity at substantially less cost.</li>
		<li>Remove the slides from the slide rack and place onto a fresh paper towel (tissue-side up). Use a Kimwipe to dry the slide. Be careful to only gently dab near (not on) the biopsy section to wick away water. Using a hydrophobic pen, draw a border around the biopsy section and place the slide tissue-side up in the humidifying chamber. 
		NOTE: Work quickly so that the tissues do not dry out before hybridization.</li>
		<li>Place the humidifying chamber into an incubator set to 50 &#176;C. Pipette 50-150 &#956;L of the staining solution directly on top of the tissue so that the rectangle made by the hydrophobic border around the tissue is filled. Be careful to not add too much solution as to overflow the hydrophobic border. Close the box gently.</li>
		<li>Incubate overnight (~16 h) at 50 &#176;C in the dark. If the incubator has a window, cover it with aluminum foil to create a dark environment. 
		NOTE: A hybridization temperature below the melting temperature of the FISH probe is required for reliable signal. 50 &#176;C is the optimal temperature for the universal 16S rRNA probe but may not be optimal for other probes.</li>
		<li>The following morning, prepare at least 500 mL of Wash buffer comprised of 0.9 M NaCl, 20 mM Tris-HCl (pH 7.2) in ddH2O and filter-sterilize into a sterile bottle with a vacuum bottle-top filter. Warm to 50-56 &#176;C in a water bath.</li>
		<li>Remove the slides from the humidifying chambers and carefully wick away any remaining hybridization solution with a Kimwipe. Place the slides in a vertical staining rack.</li>
		<li>Place the staining rack into an opaque Coplin jar containing 100 mL of pre-warmed Wash buffer for 10 min. If the Coplin jars are not opaque (e.g., glass), place them in the dark during incubation steps &#8212; perhaps under a box or in a drawer.</li>
		<li>Repeat the wash step twice with fresh Wash buffer in new Coplin jars.</li>
		<li>During the wash steps, prepare the counter-stain by diluting a 100 &#956;g/mL stock solution of Hoechst 33342 1:1,000 in wash buffer. To the same tube, add Alexa-555 wheat germ agglutinin (WGA) to a final concentration of 5&#956;g/mL and Alexa-555 Phalloidin to a final concentration of 33 nM. Store in the dark until ready for use. 
		NOTE: Hoechst, Alexa-555 WGA, and Alexa-555 Phalloidin label DNA, mucin/uroplakins, and actin, respectively and may be stored long-term in the dark per the manufacturer&#39;s instructions. Fluorescent labels used for probes and counterstains can be customized for the filter sets available for the microscope to be used.</li>
		<li>Remove the slides from the last wash and gently wick away excess wash buffer with a Kimwipe. Place the slides tissue-side up on a paper towel and add 50-150 &#956;L of counter-stain directly on top of the tissue so that the hydrophobic border is filled, but not overflowing. Cover up to four slides with a cryobox-top and incubate for 10 min at room temperature.</li>
		<li>Place the sides back into the staining rack and wash twice more in Coplin jars with fresh Wash buffer, for 10 min each.</li>
		<li>Thoroughly dry the slides after the last wash and place tissue-side up on a paper towel under a cryobox-top. Squeeze one drop of mounting media directly on top of the tissue. Gently place an appropriately sized coverslip (will depend on biopsy size) on top. Gently press out any bubbles as they will interfere with imaging and allow the cover-slipped slides to cure overnight in the dark.</li>
		<li>The next day, seal the edges of the coverslip to the slide with a light coat of clear nail polish. Allow to dry for 10 min in the dark and then store in the dark at 4 &#176;C for confocal microscopy.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>Switch on the confocal microscope and the computer software associated with the microscope per manufacturer instructions.</li>
	<li>Load the slide and visualize with the 10x objective in the blue (DAPI/Hoechst) channel. Focus carefully until nuclei are visible.</li>
	<li>Once focused, change the objective to high magnification (63x or 100x). Add oil on top of the cover slip. Refocus with the new objective, making sure that the objective lens comes in contact with oil while focusing. 
	NOTE: Use only fine focusing at high magnification (63x or 100x). Oil should only be used for an oil objective.</li>
	<li>Quickly assess each slide through the eye-piece in the green (eGFP/Alexa-488) channel to determine which slides are FISH positive and which are FISH negative. 
	NOTE: It is best if this initial assessment/scoring is done blinded by a separate individual to reduce experimental bias.</li>
	<li>To image the stained biopsies, start with a FISH positive slide and switch to the computer visualization mode. Select the 405 (Hoechst), 488 (Alexa-488), 555 (Alexa-555) channels. Set the pinhole using the longest wavelength channel, in this case 555. Find the correct focal plane for visualization of labeled bacteria in the 488 channel. Without changing the focal plane, set the gain, laser power, and offset for each channel such that the signal is not saturated, and the background is not over-corrected. Acquire the image in all three channels. 
	NOTE: Use the same settings for the 488 channel to image every slide in an experiment. The laser power may require adjustment in the 405 and 555 channels if the optimal focal plane for bacterial visualization changes between fields.</li>
	<li>Repeat on additional fields until acquiring images of the entire epithelial surface. 
	NOTE: You may have to change the focal plane slightly to visualize labeled bacteria in different fields, but never change the gain, laser power, or offset for the 488 channel between fields. If working on a confocal microscope, it may be informative to capture a Z-stack so that the three-dimensional localization of the bacteria within the tissue may be analyzed.</li>
	<li>Process images and quantify labeled bacteria within or associated with the tissue using ImageJ or similar software. Minimal processing of the images is recommended (e.g., splitting/merging channels and converting into image files), although background correction may be performed if necessary. All corrections or other alterations should remain consistent between images.</li>
</ol>

<ol>
	<li>Abraham, S. N., Miao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nature+of+immune+responses+to+urinary+tract+infections.">The nature of immune responses to urinary tract infections.</a> Nature Reviews Immunology. 15 (10), 655-663 (2015).</li><li>Wolfe, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+uncultivated+bacteria+in+the+adult+female+bladder.">Evidence of uncultivated bacteria in the adult female bladder.</a> Journal of Clinical Microbiology. 50 (4), 1376-1383 (2012).</li><li>Grist, M., Chakraborty, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+mucin+layer+in+the+urinary+bladder.">Identification of a mucin layer in the urinary bladder.</a> Urology. 44 (1), 26-33 (1994).</li><li>Wu, X. R., Kong, X. P., Pellicer, A., Kreibich, G., Sun, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uroplakins+in+urothelial+biology,+function,+and+disease.">Uroplakins in urothelial biology, function, and disease.</a> Kidney International. 75 (11), 1153-1165 (2009).</li><li>Ingersoll, M. A., Albert, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+infection+to+immunotherapy:+host+immune+responses+to+bacteria+at+the+bladder+mucosa.">From infection to immunotherapy: host immune responses to bacteria at the bladder mucosa.</a> Mucosal Immunology. 6 (6), 1041-1053 (2013).</li><li>Flores-Mireles, A. L., Walker, J. N., Caparon, M., Hultgren, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urinary+tract+infections:+epidemiology,+mechanisms+of+infection+and+treatment+options.">Urinary tract infections: epidemiology, mechanisms of infection and treatment options.</a> Nature Reviews Microbiology. 13 (5), 269-284 (2015).</li><li>Lewis, A. J., Richards, A. C., Mulvey, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+Host+Cells+and+Tissues+by+Uropathogenic+Bacteria.">Invasion of Host Cells and Tissues by Uropathogenic Bacteria.</a> Microbiology Spectrum. 4 (6), (2016).</li><li>Horsley, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enterococcus+faecalis+subverts+and+invades+the+host+urothelium+in+patients+with+chronic+urinary+tract+infection.">Enterococcus faecalis subverts and invades the host urothelium in patients with chronic urinary tract infection.</a> PLoS One. 8 (12), (2013).</li><li>Rosen, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+an+intracellular+bacterial+community+pathway+in+Klebsiella+pneumoniae+urinary+tract+infection+and+the+effects+of+FimK+on+type+1+pilus+expression.">Utilization of an intracellular bacterial community pathway in Klebsiella pneumoniae urinary tract infection and the effects of FimK on type 1 pilus expression.</a> Infection and Immunity. 76 (7), 3337-3345 (2008).</li><li>Rosen, D. A., Hooton, T. M., Stamm, W. E., Humphrey, P. A., Hultgren, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+intracellular+bacterial+communities+in+human+urinary+tract+infection.">Detection of intracellular bacterial communities in human urinary tract infection.</a> PLoS Med. 4 (12), e329 (2007).</li><li>Hussain, S. A., Alhalabi, F., Zimmern, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+efficacy+of+fulguration+of+trigonitis+for+recurrent+urinary+tract+infections+in+women.">Long-term efficacy of fulguration of trigonitis for recurrent urinary tract infections in women.</a> Urological Science. 26 (3), 197-201 (2015).</li><li>De Nisco, N. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Detection+of+Tissue-Resident+Bacteria+and+Chronic+Inflammation+in+the+Bladder+Wall+of+Postmenopausal+Women+with+Recurrent+Urinary+Tract+Infection.">Direct Detection of Tissue-Resident Bacteria and Chronic Inflammation in the Bladder Wall of Postmenopausal Women with Recurrent Urinary Tract Infection.</a> Journal of Molecular Biology. , (2019).</li><li>Amann, R. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+of+16S+rRNA-targeted+oligonucleotide+probes+with+flow+cytometry+for+analyzing+mixed+microbial+populations.">Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations.</a> Applied and Environmental Microbiology. 56 (6), 1919-1925 (1990).</li><li>Vaishnava, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antibacterial+lectin+RegIIIgamma+promotes+the+spatial+segregation+of+microbiota+and+host+in+the+intestine.">The antibacterial lectin RegIIIgamma promotes the spatial segregation of microbiota and host in the intestine.</a> Science. 334 (6053), 255-258 (2011).</li><li>Greuter, D., Loy, A., Horn, M., Rattei, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=probeBase--an+online+resource+for+rRNA-targeted+oligonucleotide+probes+and+primers:+new+features+2016.">probeBase--an online resource for rRNA-targeted oligonucleotide probes and primers: new features 2016.</a> Nucleic Acids Research. 44 (D1), D586-D589 (2016).</li><li>Monici, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+and+tissue+autofluorescence+research+and+diagnostic+applications.">Cell and tissue autofluorescence research and diagnostic applications.</a> Biotechnology Annual Review. 11, 227-256 (2005).</li><li>Mysorekar, I. U., Hultgren, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+uropathogenic+Escherichia+coli+persistence+and+eradication+from+the+urinary+tract.">Mechanisms of uropathogenic Escherichia coli persistence and eradication from the urinary tract.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (38), 14170-14175 (2006).</li><li>Frickmann, H., Lakner, A., Essig, A., Poppert, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+identification+of+yeast+by+fluorescence+in+situ+hybridisation+from+broth+and+blood+cultures.">Rapid identification of yeast by fluorescence in situ hybridisation from broth and blood cultures.</a> Mycoses. 55 (6), 521-531 (2012).</li><li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraffin+embedding+tissue+samples+for+sectioning.">Paraffin embedding tissue samples for sectioning.</a> Cold Spring Harbor Protocols. 2008, (2008).</li><li>Sun, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sudan+black+B+reduces+autofluorescence+in+murine+renal+tissue.">Sudan black B reduces autofluorescence in murine renal tissue.</a> Archives of Pathology &amp; Laboratory Medicine. 135 (10), 1335-1342 (2011).</li><li>Ludwig, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ARB:+a+software+environment+for+sequence+data.">ARB: a software environment for sequence data.</a> Nucleic Acids Research. 32 (4), 1363-1371 (2004).</li><li>Jaziri, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PhylOPDb:+a+16S+rRNA+oligonucleotide+probe+database+for+prokaryotic+identification.">PhylOPDb: a 16S rRNA oligonucleotide probe database for prokaryotic identification.</a> Database (Oxford). , (2014).</li></ol>

The authors have nothing to disclose.

Here, we describe a protocol for the detection of tissue-associated bacteria in human bladder biopsies by 16S rRNA FlSH. This protocol can be easily adapted for biopsies taken from other tissues, such as the gastrointestinal tract or skin, and can be extended to tissues harvested from a variety of mammalian model organisms. The protocol described here can also be adapted to for the use of multiple fixation (e.g., formalin, ethanol, methacarn) and tissue preparation techniques (e.g., paraffin or resin embedded, and cryopreserved tissues). The double-labeled universal 16S rRNA probe allows for the unbiased detection of all bacterial species present within tissue and can provide valuable insight into how pathogens and the microbiota spatially interact with mucosal surfaces in disease and healthy states. Using resources such as probeBase, PhylOPDb or the PROBE_DESIGN tool of the ARB software package for the selection or design of species or genera-specific 16S or 23S rRNA probes, this protocol can be adapted for the detection of specific bacterial species or genera within tissue15,21,22. An important future direction for this method is multiplexing using species- or genera-specific probes labeled with different, discrete fluorophores for evaluation of microbial diversity within the bladder mucosa.
The primary limitation of this method for use on human specimens is the availability of biopsied tissue. Institutional Review Board approval and informed patient consent are required to obtain biopsies and direct collaboration with the clinician performing the procedure is necessary for optimal sample collection and access to patient metadata. The CEFT procedure itself destroys the bladder epithelium so we were able to justify biopsy of these areas before the procedure. A fume hood or appropriately fitted biosafety cabinet is required for this protocol due to the use of toxic xylenes in the deparaffinization step and the need to maintain a sterile environment throughout the procedure. A fluorescent microscope, preferably confocal, with a 63x or a 100x objective and appropriate filter sets for visualization of Hoechst, Alexa-555, and Alexa-488 is required for this protocol. The representative results depicted in Figure 1 were imaged using a laser scanning confocal microscope. Similar laser scanning microscopes should produce comparable images. This protocol is limited by its ability to only detect tissue-associated bacteria and not, for example, fungi. Probes specific the fungal 18S or 28S rRNA must be used to identify fungal pathogens within tissue18.
Critical steps to this protocol include maintaining a sterile environment throughout the procedure and ensuring that the tissue does not dry out between hybridization and staining steps. If the tissue dries out during the procedure, the signal may be dampened or the tissue may fall off of the slide during a wash step. It is also critical to always use two serial sections for this protocol &#8212; one for the 16S rRNA probe and one for the scramble probe. Without this control, it may be very difficult to distinguish false positives and the data obtained may not be useful or informative. If this protocol is being adapted for use with a probe other than the universal 16S rRNA probe, care must be taken to select an appropriate hybridization temperature, approximately 5 &#176;C lower than the predicted melting temperature of the probe. To maintain signal intensity, the tissue must not be exposed to light for long periods of time after the probe has been added and must not be overexposed during microscopy. Lastly, during microscopy the same settings for the channel corresponding to the fluorophore conjugated to the FISH probe must be kept consistent between the experimental (16S rRNA probe) and control (scramble probe) slides. Visualizing the spatial relationship of bacteria within mucosal surfaces of patient-derived tissues is critical to understanding and building clinically relevant hypotheses about the host-pathogen interactions underlying infectious disease.

The urinary tract, consisting of the urethra, bladder, ureters and kidneys is constantly exposed to bacteria that comprise the urinary microbiome as well as invading uropathogens, like uropathogenic E. coli (UPEC), from the gastrointestinal tract1,2. A layer of hydrated mucus consisting of glycosaminoglycans and an impermeable plaque of glycosylated uroplakin proteins expressed on the surface of the superficial cells form a barrier that routinely protects the bladder epithelium from invasion by adherent bacteria3,4. During urinary tract infection (UTI), these barriers are disturbed or destroyed, facilitating attachment to and invasion of the bladder epithelium by uropathogenic bacteria5,6. Work in murine models has revealed that many uropathogenic bacteria including UPEC, Klebsiella pneumoniae, and Enterococcus faecalis can form replicative intracellular communities (IBCs) within the cytoplasm of superficial cells and quiescent intracellular reservoirs (QIRs) within transitional epithelial cells7,8,9. Although UPEC has been identified within shed epithelial cells from human UTI patients, the interaction of uropathogens with the bladder mucosa in humans had not been previously visualized10.
We adapted a common technique, fluorescence in situ hybridization (FISH), to detect bacteria within the mucosa of bladder biopsies obtained from postmenopausal patients undergoing cystoscopy with electrofulguration of trigonitis (CEFT) for the advanced management of antibiotic-refractory recurrent UTI11. Using a universal probe for 16S rRNA, we were able to objectively detect bacterial species associated with the bladder mucosa of recurrent UTI patients and determine their position within the bladder wall12. The universal 16S rRNA nucleotide probe was previously designed to target a conserved region of the bacterial 16S rRNA13, which corresponds to positions 388-355 of the E. coli 16s rRNA. The 16S rRNA and scramble probe sequences have been previously validated and published for use in the mouse gastrointestinal tract14,15. The sequences and properties of the probes are described in Table 1. It is essential to use two sequential sections in this protocol, one for the 16S rRNA probe and one for the scramble probe, to be able to distinguish between true and background signal as the bladder epithelium, collagen and elastin exhibit autofluorescence16. In this protocol, the 16S rRNA and scramble probes were designed with fluorescent Alexa Fluor 488 labels on both the 3&#39; and 5&#39; termini via N-hydroxysuccinimide (NHS) ester linkages to increase fluorescent signal.
Although this protocol was developed for use on human bladder biopsy sections, it can readily be adapted for use on paraffin-embedded sections from any tissue where bacteria are believed to reside. Unlike immunohistochemistry experiments that target specific antigens (e.g., lipopolysaccharide) on the bacterial surface, this method requires no prior knowledge of antigens expressed by the tissue-associated bacteria10,17. Use of the universal 16S rRNA probe allows the unbiased detection of all bacterial species within the sample but does not allow determination of their identity. To determine the identify of detected bacteria, species or genus-specific 16S or 23S rRNA probes must be used. This protocol will also not detect fungal pathogens, such as Candida albicans, associated with host tissue. For detection of fungal pathogens, 28S or 18S rRNA probes must be used18.

<table><tbody><tr><td>Alexa-555 Phalloidin</td><td>Invitrogen</td><td>A34055</td><td>Staining Actin</td></tr><tr><td>Alexa-555 Wheat Germ Agglutinin (WGA)</td><td>Invitrogen</td><td>W32464</td><td>Staining Mucin</td></tr><tr><td>Bottle top filters</td><td>Fisher Scientific</td><td>09-741-07</td><td>Sterilization</td></tr><tr><td>Coplin Jar</td><td>Simport</td><td>M900-12W</td><td>Deparaffinization/washing</td></tr><tr><td>Coverslips</td><td>Fisher Scientific</td><td>12-548-5M</td><td>Microscopy</td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>04-355-224</td><td>Rehydration</td></tr><tr><td>Ethylenediaminetetraacetic Acid, Disodium Salt Dihydrate</td><td>Fisher Scientific</td><td>S311-500</td><td>TE</td></tr><tr><td>Frosted Slides</td><td>Thermo Fisher Scientific</td><td>12-550-343</td><td></td></tr><tr><td>Hoechst 33342, Trihydrochloride, trihydrate</td><td>Invitrogen</td><td>H21492</td><td>Staining Nucleus</td></tr><tr><td>Hydrophobic marker</td><td>Vector Laboratories</td><td>H-4000</td><td>Hydrophobic barrier</td></tr><tr><td>Kimwipes</td><td>Fisher Scientific</td><td>06-666-11</td><td></td></tr><tr><td>Oil Immersol 518 F</td><td>Fisher Scientific</td><td>12-624-66A</td><td>Microscopy</td></tr><tr><td>Paraformaldehyde (16%)</td><td>Thermo Scientific</td><td>TJ274997</td><td>Fixation</td></tr><tr><td>ProLong Gold antifade reagent</td><td>Invitrogen</td><td>P36934</td><td>Mounting medium</td></tr><tr><td>Sodium Chloride</td><td>Fisher Scientific</td><td>BP358-10</td><td>Hybridization buffer/PBS</td></tr><tr><td>Sodium Dodecyl Sulphate</td><td>Fisher Scientific</td><td>BP166-500</td><td>Hybridization buffer</td></tr><tr><td>Sodium Phosphate Dibasic Hepahydrate</td><td>Fisher Scientific</td><td>S373-500</td><td>PBS</td></tr><tr><td>Sodium Phosphate Monobasic Monohydrate</td><td>Fisher Scientific</td><td>S369-500</td><td>PBS</td></tr><tr><td>Syringe</td><td>VWR</td><td>75486-756</td><td>Sterilization</td></tr><tr><td>Tris-HCl</td><td>Fisher Scientific</td><td>BP152-5</td><td>TE/Hybridization buffer</td></tr><tr><td>Xylene</td><td>Fisher Chemical</td><td>X3P-1GAL</td><td>Deparaffinization</td></tr><tr><td>0.22 &amp;mu;m syringe filter</td><td>Fisher Scientific</td><td>09-754-29</td><td>Sterilization</td></tr></tbody></table>

detection of tissue-resident bacteria in bladder biopsies by 16s rrna fluorescence in situ hybridization

Visualization of the interaction of bacteria with host mucosal surfaces and tissues can provide valuable insight into mechanisms of pathogenesis. While visualization of bacterial pathogens in animal models of infection can rely on bacterial strains engineered to express fluorescent proteins such as GFP, visualization of bacteria within the mucosa of biopsies or tissue obtained from human patients requires an unbiased method. Here, we describe an efficient method for the detection of tissue-associated bacteria in human biopsy sections. This method utilizes fluorescent in situ hybridization (FISH) with a fluorescently labeled universal oligonucleotide probe for 16S rRNA to label tissue-associated bacteria within bladder biopsy sections acquired from patients suffering from recurrent urinary tract infection. Through use of a universal 16S rRNA probe, bacteria can be detected without prior knowledge of species, genera, or biochemical characteristics, such as lipopolysaccharide (LPS), that would be required for detection by immunofluorescence experiments. We describe a complete protocol for 16S rRNA FISH from biopsy fixation to imaging by confocal microscopy. This protocol can be adapted for use in almost any type of tissue and represents a powerful tool for the unbiased visualization of clinically-relevant bacterial-host interactions in patient tissue. Furthermore, using species or genera-specific probes, this protocol can be adapted for the detection of specific bacterial pathogens within patient tissue.

The protocol has been optimized for the unbiased detection of bacteria associated with the bladder mucosa in paraffin-embedded bladder biopsy sections. Figure 1 depicts representative confocal micrographs from an experiment using this protocol on sections of bladder biopsies obtained from women with recurrent urinary tract infection. Two serial sections were hybridized with either the universal 16S rRNA (upper panels) or scramble (lower panels) probes. Images from the same region of the tissue were taken and bacteria (green) are clearly visible in the tissue hybridized with the 16S rRNA probes and not with the scramble probe. Figure 2 represents a false positive result. Signal corresponding to autofluorescent collagen or elastin is detected in the 405 and 488 channels in both the 16S rRNA and scramble probe-hybridized biopsy sections highlighting the importance of always using a scramble probe control.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Probe</td>
			<td>Sequence</td>
			<td>Tm</td>
			<td>Fluorophore</td>
			<td>Linkage</td>
		</tr>
		<tr>
			<td>Universal 16S rRNA&#160;</td>
			<td>5&#39;-GCTGCCTCCC 
			GTAGGAGT-3&#39;</td>
			<td>54.9</td>
			<td>Alexa-488 (5&#39; and 3&#39;)</td>
			<td>NHS Ester</td>
		</tr>
		<tr>
			<td>Scramble</td>
			<td>5&#39;-ACTCCTACGG 
			GAGGCAGC-3&#39;</td>
			<td>NA</td>
			<td>Alexa-488 (5&#39; and 3&#39;)</td>
			<td>NHS Ester</td>
		</tr>
	</tbody>
</table>
Table 1: FISH probe sequences and characteristics. Tm indicates melting temperature and NHS is an abbreviation for N-hydroxysuccinimide.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60458/60458fig1.jpg" /> 
Figure 1: Representative confocal micrographs of FISH of universal 16S rRNA and scramble probe in a human bladder biopsy. Actin and Mucin are labeled in red, cellular nuclei are labeled in blue, and bacteria in green. Tissue-associated bacteria are only detected with the 16SrRNA probe and not the scramble. Images taken at 63x magnification. Scale bar = 20 &#956;m. <a href="https://www.jove.com/files/ftp_upload/60458/60458fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60458/60458fig2.jpg" /> 
Figure 2: Representative confocal micrographs of false-positive green autofluorescence in a human bladder biopsy. Actin and mucin are labeled in red, cellular nuclei are labeled in blue, and bacteria and autofluorescent components of the extracellular matrix (e.g., collagen and elastin) are green. Green fluorescence is observed with both the 16S rRNA and scramble probes indicating a false-positive result. Images are taken at 63x magnification. Scale bar = 20 &#956;m. <a href="https://www.jove.com/files/ftp_upload/60458/60458fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Detection of Tissue-resident Bacteria in Bladder Biopsies by 16S rRNA Fluorescence In Situ Hybridization at JoVE.com

Detection of Tissue-resident Bacteria in Bladder Biopsies by 16S rRNA Fluorescence In Situ Hybridization

This protocol is for the unbiased detection of tissue-associated bacteria in patient biopsies by 16S rRNA in situ hybridization and confocal microscopy.

Visualization of the interaction of bacteria with host mucosal surfaces and tissues can provide valuable insight into mechanisms of pathogenesis. While ...

detection-tissue-resident-bacteria-bladder-biopsies-16s-rrna

Research

JoVE Journal

Biology

9.2K Views.  University of Texas at Dallas. This protocol is for the unbiased detection of tissue-associated bacteria in patient biopsies by 16S rRNA in situ hybridization and confocal microscopy.

Video: Detection of Tissue-resident Bacteria in Bladder Biopsies by 16S rRNA Fluorescence In Situ Hybridization

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

Immunology and Infection

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

Genetics

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

Neuroscience

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been reported using various methods, including electron microscopy, immunohistochemistry or immunofluorescence using bacteria-specific antibodies, and fluorescent in situ hybridization (FISH) using a fluorescence-labeled oligonucleotide probe. Nevertheless, these methods are not widely used due to technical limitation or difficulties. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled DNA probes has been introduced. The paraffin-embedded tissues are the most common form of biopsy tissues available from pathology banks. Bacteria can be detected either in a species-specific or universal manner. Bacterial signals are detected as either discrete forms (coccus, rod, fusiform, and hairy form) of bacteria or dispersed forms. The technique allows other histological information to be obtained: the epithelia, connective tissue, inflammatory infiltrates, and blood vessels are well distinguished. This method can be used to study the role of bacteria in various diseases, such as periodontitis, cancers, and inflammatory immune diseases.

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled 16S rRNA-targeting DNA probes has been described. This protocol can be applied to study the role of bacteria in various diseases such as periodontitis, cancers, and inflammatory immune diseases.

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been ...

Enhancing FISH Analysis for Oncogenic Drivers in Cancer Tissue Samples

Robust Detection of Gene Amplification in Formalin-Fixed Paraffin-Embedded Samples by Fluorescence In Situ Hybridization

Focal gene amplification, such as extrachromosomal DNA (ecDNA), plays an important role in cancer development and therapy resistance. While sequencing-based methodologies enable an unbiased identification of ecDNA, cytogenetic-based techniques, such as fluorescence in situ hybridization (FISH), remain time and cost-effective for identifying ecDNA in clinical specimens. The application of FISH in formalin-fixed paraffin-embedded (FFPE) tissue samples offers a unique avenue for detecting amplified genes, particularly when viable specimens are not available for karyotype examination. However, there is a lack of consensus procedures for this technique. This protocol provides comprehensive, fully optimized, step-by-step instructions for conducting FISH to detect gene amplification, including ecDNA, in FFPE tissue samples which present unique challenges that this protocol aims to overcome and standardize. By following this protocol, researchers can reproducibly acquire high-quality imaging data to assess gene amplification.

Author Spotlight: FISH as a Tool for Precise Gene Amplification Assessment in Cancer Specimens

This protocol provides a reproducible method to visualize gene amplification in formalin-fixed paraffin-embedded (FFPE) tissue specimens.

Focal gene amplification, such as extrachromosomal DNA (ecDNA), plays an important role in cancer development and therapy resistance. While ...

Cancer Research

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.

This protocol describes a simple method of isolating single, infected-bladder epithelial cells from a murine model of urinary tract infection.

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...

Longitudinal Follow-Up of Urinary Tract Infections and Their Treatment in Mice using Bioluminescence Imaging

Urinary tract infections (UTI) rank among the most common bacterial infections in humans and are routinely treated with empirical antibiotics. However, due to increasing microbial resistance, the efficacy of the most used antibiotics has declined. To find alternative treatment options, there is a great need for a better understanding of the UTI pathogenesis and the mechanisms that determine UTI susceptibility. In order to investigate this in an animal model, a reproducible, non-invasive assay to study the course of UTI is indispensable.
For years, the gold standard for the enumeration of bacterial load has been the determination of Colony Forming Units (CFU) for a particular sample volume. This technique requires post-mortem organ homogenates and serial dilutions, limiting data output and reproducibility. As an alternative, bioluminescence imaging (BLI) is gaining popularity to determine the bacterial load. Labeling pathogens with a lux operon allow for the sensitive detection and quantification in a non-invasive manner, thereby enabling longitudinal follow-up. So far, the adoption of BLI in UTI research remains limited.
This manuscript describes the practical implementation of BLI in a mouse urinary tract infection model. Here, a step-by-step guide for culturing bacteria, intravesical instillation and imaging is provided. The in vivo correlation with CFU is examined and a proof-of-concept is provided by comparing the bacterial load of untreated infected animals with antibiotic-treated animals. Furthermore, the advantages, limitations, and considerations specific to the implementation of BLI in an in vivo UTI model are discussed. The implementation of BLI in the UTI research field will greatly facilitate research on the pathogenesis of UTI and the discovery of new ways to prevent and treat UTI.

This manuscript describes the intravesical administration of uropathogenic bacteria with a lux operon to induce a urinary tract infection in mice and subsequent longitudinal in vivo analysis of the bacterial load using bioluminescence imaging.

Urinary tract infections (UTI) rank among the most common bacterial infections in humans and are routinely treated with empirical antibiotics. However, ...

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota and the vertebrate gut. The spatial landscape of the gut microbiota is tightly controlled by physical features - intestinal mucus, crypts, and folds - and is affected by host-controlled properties such as pH, oxygen availability, and immune factors. These properties limit the ability of commensal microbes and pathogens alike to colonize the gut stably. At the micron-scale, microbial organization determines the close-range interactions between different microbes as well as the interactions between microbes and their host. These interactions then affect large-scale organ function and host health.
This protocol enables the visualization of the gut microbiota spatial organization from distances between cells to organ-wide scales. The method is based on fixing gut tissues while preserving intestinal structure and mucus properties. The fixed samples are then embedded, sectioned, and stained to highlight specific bacterial species through fluorescence in situ hybridization (FISH). Host features, such as mucus and host cell components, are labeled with fluorescently labeled lectins. Finally, the stained sections are imaged using a confocal microscope utilizing tile-scan imaging at high magnification to bridge the micron to centimeter length scales. This type of imaging can be applied to intestinal sections from animal models and biopsies from human tissues to determine the biogeography of the microbiota in the gut in health and disease.

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

This streamlined protocol details a workflow to detect and image bacteria in complex tissue samples, from fixing the tissue to staining microbes with fluorescent in situ hybridization.

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota ...

Detection of Tissue-resident Bacteria in Bladder Biopsies by 16S rRNA Fluorescence In Situ Hybridization

Summary

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Chapters in this video

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

Robust Detection of Gene Amplification in Formalin-Fixed Paraffin-Embedded Samples by Fluorescence In Situ Hybridization

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Longitudinal Follow-Up of Urinary Tract Infections and Their Treatment in Mice using Bioluminescence Imaging

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging