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>

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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 micron 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

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

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 ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ ...

Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing

The human intestinal microbiome plays a central role in protecting cells from injury, in processing energy and nutrients, and in promoting immunity. Deviations from what is considered a healthy microbiota composition (dysbiosis) may impair vital functions leading to pathologic conditions. Recent and ongoing research efforts have been directed toward the characterization of associations between microbial composition and human health and disease.
Advances in high-throughput sequencing technologies enable characterization of the gut microbial composition. These methods include 16S rRNA-amplicon sequencing and shotgun sequencing. 16S rRNA-amplicon sequencing is used to profile taxonomical composition, while shotgun sequencing provides additional information about gene predictions and functional annotation. An advantage in using a targeted sequencing method of the 16S rRNA gene variable region is its substantially lower cost compared to shotgun sequencing. Sequence differences in the 16S rRNA gene are used as a microbial fingerprint to identify and quantify different taxa within an individual sample.
Major international efforts have enlisted standards for 16S rRNA-amplicon sequencing. However, several studies report a common source of variation caused by batch effect. To minimize this effect, uniformed protocols for sample collection, processing, and sequencing must be implemented. This protocol proposes the integration of broadly used protocols starting from fecal sample collection to data analyses. This protocol includes a column-free, direct-PCR approach that enables simultaneous handling and DNA extraction of large numbers of fecal samples, along with PCR amplification of the V4 region. In addition, the protocol describes the analysis pipeline and provides a script using the latest version of QIIME (QIIME 2 version 2017.7.0 and DADA2). This step-by-step protocol is aimed to guide those interested in initiating the use of 16S rRNA-amplicon sequencing in a robust, reproductive, easy to use, detailed way.

This manuscript describes a detailed standardized protocol of high-throughput 16S rRNA-amplicon sequencing. The protocol introduces an integrated, uniformed, feasible, and inexpensive protocol starting from fecal sample collection through data analyses. This protocol enables analysis of large numbers of samples with rigorous standards and several controls.

The human intestinal microbiome plays a central role in protecting cells from injury, in processing energy and nutrients, and in promoting immunity. ...

Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective and widely available vaccines are lacking for a majority of these diseases, necessitating the development of novel disease mitigation strategies. To this end, a promising avenue of disease control involves targeting the vector microbiome, the community of microbes inhabiting the vector. The vector microbiome plays a pivotal role in pathogen dynamics, and manipulations of the microbiome have led to reduced vector abundance or pathogen transmission for a handful of vector-borne diseases. However, translating these findings into disease control applications requires a thorough understanding of vector microbial ecology, historically limited by insufficient technology in this field. The advent of next-generation sequencing approaches has enabled rapid, highly parallel sequencing of diverse microbial communities. Targeting the highly-conserved 16S rRNA gene has facilitated characterizations of microbes present within vectors under varying ecological and experimental conditions. This technique involves amplification of the 16S rRNA gene, sample barcoding via PCR, loading samples onto a flow cell for sequencing, and bioinformatics approaches to match sequence data with phylogenetic information. Species or genus-level identification for a high number of replicates can typically be achieved through this approach, thus circumventing challenges of low detection, resolution, and output from traditional culturing, microscopy, or histological staining techniques. Therefore, this method is well-suited for characterizing vector microbes under diverse conditions but cannot currently provide information on microbial function, location within the vector, or response to antibiotic treatment. Overall, 16S next-generation sequencing is a powerful technique for better understanding the identity and role of vector microbes in disease dynamics.

Here we present a next-generation sequencing protocol for 16S rRNA sequencing which enables identification and characterization of microbial communities within vectors. This method involves DNA extraction, amplification and barcoding of samples through PCR, sequencing on a flow-cell, and bioinformatics to match sequence data to phylogenetic information.

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective ...