visualization of bacteria in bladder biopsy sections via fluorescence in situ hybridization

Visualization of Bacteria in Bladder Biopsy Sections via Fluorescence In Situ Hybridization

Prepare a humidifying chamber for each probe by adding a soaked, crumpled, delicate task wipe and sterile water to the reservoir of a P1000 tip box. Place the tip holder cartridge on top, as this is where the slides will sit. When the final wash is complete, remove the slide from the slide rack, and place them onto a fresh paper towel, tissue-side up. Use a delicate task wipe to dry the slide, being careful to only gently dab near but not on the biopsy section to wick away the water. Using a hydrophobic pen, draw a border around the biopsy section, and place the slide tissue-side up in the humidifying chamber.
Next, place the humidifying chamber into an incubator set to 50 degrees Celsius. Pipette between 50 and 150 microliters of the staining solution directly on top of the tissue so that the rectangle made by the hydrophobic border around the tissue is filled, and close the box gently. Incubate overnight at 50 degrees Celsius in the dark. If the incubator has a window, cover it with aluminum foil to create a dark environment.
The next morning, remove the slides from the humidifying chambers, and quickly wick away any remaining hybridization solution with a delicate task wipe. Then, place the slides into a vertical staining rack. Place the staining rack into an aluminum foil-wrapped coplin jar containing 100ml of the pre-warmed wash buffer for 10 minutes. Repeat this wash step two more times with fresh wash buffer in new coplin jars.
During these wash steps, prepare the counterstain by diluting a stock solution of Hoechst 33342 in wash buffer at a ratio of 1 to 1,000. To the same tube, add Alexa-555 wheat germ agglutinin to a final concentration of five micrograms per milliliter and Alexa-555 phalloidin to a final concentration of 33 nanomolar. Store in the dark until ready to use.
When the final wash is complete, remove the slides from the coplin jar, and gently wick away any excess wash buffer with a delicate task wipe. Place the slides tissue-side up on a paper towel, and add between 50 and 150 microliters of counterstain 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 at room temperature for 10 minutes.
After this, place the slides back into the staining rack, and wash them twice more in coplin jars containing fresh wash buffer, with each wash lasting 10 minutes. Then, thoroughly dry the slides, and place them tissue-side up on a paper towel under a cryobox top. Squeeze one drop of mounting media directly on top of the tissue, and gently place an appropriately-sized coverslip on top. Gently, press out any bubbles, and allow the cover-slipped slides to cure overnight in the dark. The next day, seal the edges of the coverslips to the slide with a light coat of clear nail polish, and let dry in the dark for 10 minutes, before storing them in the dark at 4 degrees Celsius.
When ready to image the stained biopsies, switch on the confocal microscope and the software associated with the microscope. Start with a FISH-positive slide and switch to the computer visualization mode. Select the channels for 405, 488, and 555. Set the pinhole using the longest wavelength channel, which in this case is 555. Then, find the current 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 overcorrected. Acquire the image in all three channels.

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Tissue Preparation for Fixation and Paraffin Embedding
NOTE:&#160;Biopsies were taken from consenting women undergoing cystoscopy with electro-fulguration of trigonitis for the advanced management of recurrent urinary tract infection (rUTI). rUTI is defined as 3 UTIs in 12 months. Biopsy collection was performed in the operating room while the patient was under anesthesia after obtaining informed patient consent. All samples were coded and de-identified before experimentation.
<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).&#160;&#160; 
	NOTE:&#160;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:&#160;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 the fixative by pipetting and replace it with 1.5 mL of sterile 1x phosphate-buffered saline (PBS). Keep the samples at 4 &#176;C overnight or immediately perform tissue processing and paraffin embedding.</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 ribosomal RNA (16S rRNA) fluorescence in situ hybridization (FISH) protocol. 
	NOTE:&#160;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:&#160;Two slides per biopsy are required. One slide is needed for the universal 16S rRNA probe and one slide for a control probe with a scrambled sequence. This is important for distinguishing the true signal from the background signal during microscopy since the bladder epithelium is auto-fluorescent in multiple channels. In addition to the scramble probe, blocking with 0.1% Sudan Black B prior to mounting may reduce background autofluorescence inherent in the tissue.
<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 (hydroxymethyl) aminomethane-hydrochloride (Tris-HCl, pH 7.2), 0.1% sodium dodecyl sulfate (SDS) in 10 mL of sterile-filtered water.&#160; 
		NOTE:&#160;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 the 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.&#160;&#160;&#160;&#160; 
		NOTE:&#160;Do not dissolve fluorescently labeled probes in water. Buffering is required to prevent hydrolysis of the N-hydroxysuccinimide (NHS) ester bond conjugating the fluorophore to the nucleotide probe.</li>
		<li>Clean five Coplin jars with 70% ethanol (EtOH), allow to dry, label as follows: xylenes I, xylenes II, 95% EtOH, 90% EtOH, double-distilled water (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:&#160;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:&#160;During this stage, warm the 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 a hybridization buffer to create the staining solution. Prepare 150 &#956;L of staining solution per slide.&#160;&#160; 
		NOTE:&#160;Protect the staining solution from light by wrapping the tubes in aluminum foil and storing them in a drawer. When working with fluorescently labeled probes on the benchtop, consider turning off overhead lights when able. Probes diluted in the 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 in the reservoir of a P1000 tip box. Place the tip holder cartridge on top - this is where the slides will sit. 
		NOTE:&#160;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 them 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.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
		NOTE:&#160;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 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:&#160;A hybridization temperature below the melting temperature of the FISH probe is required for a 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 sodium chloride (NaCl), and 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 the 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.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
		NOTE:&#160;Hoechst, Alexa-555 WGA, and Alexa-555 Phalloidin label deoxyribonucleic acid (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 cryo box 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 cryo box 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:&#160;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 the manufacturer&#39;s instructions.</li>
	<li>Load the slide and visualize with the 10x objective in the blue (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 coverslip. Refocus with the new objective, making sure that the objective lens comes in contact with oil while focusing. 
	NOTE:&#160;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 (enhanced green fluorescent protein, eGFP/Alexa-488) channel to determine which slides are FISH positive and which are FISH negative. 
	NOTE:&#160;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), and 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.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE:&#160;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>
</ol>

<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>

Source: Neugent, M. L. et al., <a href="https://app.jove.com/v/60458">Detection of Tissue-resident Bacteria in Bladder Biopsies by 16S rRNA Fluorescence In Situ Hybridization.</a>&#160;J. Vis. Exp. (2019)
This video demonstrates the visualization of bacteria in deparaffinized bladder tissue from a urinary tract-infected patient. Fluorescently-labeled probe hybridized with bacterial 16s ribosomal RNAs reveals the tissue-associated bacteria and their distribution, providing valuable spatial insights into their density within the sample.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Tissue Preparation for Fixation ...

Begin with a glass slide carrying a deparaffinized and rehydrated infected urinary bladder biopsy tissue section containing uropathogenic bacteria.
Introduce fluorescently-labeled universal probes &#8212; a complementary single-stranded oligonucleotide specific to the conserved region of bacterial 16s ribosomal RNA, or 16s-rRNA.
Incubate the slide in the dark at an elevated temperature for optimum hybridization.
During incubation, the probes exclusively hybridize to the conserved region on bacterial 16s-rRNA, imparting green fluorescence to the bacteria.&#160;
Post-hybridization, wash with a pre-warmed buffer to remove the unbound probes.
Stain the tissue section with Hoechst dye and fluorophore-labeled cellular protein markers.
Hoechst molecules bind to the DNA, staining the nuclei blue, while fluorophore-labeled markers interact with specific cell components such as mucin and actin filaments, imparting red fluorescence in the biopsy section.
Wash the slide and add mounting media to the tissue section.
Under a confocal microscope, green fluorescence within the red fluorescence-carrying bladder biopsy section confirms the tissue-associated bacteria.

60 ビュー. 蛍光In Situハイブリダイゼーションによる膀胱生検切片の細菌の可視化

ビデオ: 蛍光In Situハイブリダイゼーションによる膀胱生検切片の細菌の可視化 - 実験

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.

16S rRNAのをターゲットジゴキシン標識DNAプローブを用いたパラフィン包埋組織内細菌のその場検出で

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

JoVE Journal

免疫・感染学

RNA Fluorescence in situ Hybridization (FISH) to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as microsporidia and viruses. Because of the similarities between Caenorhabditis elegans and mammalian intestinal cells, as well as the power of the C. elegans system, this host has emerged as a model system to study host intestine-microbe interactions in vivo. While it is possible to observe some aspects of these interactions with bright-field microscopy, it is difficult to accurately classify microbes and characterize the extent of colonization or infection without more precise tools. RNA fluorescence in situ hybridization (FISH) can be used as a tool to identify and visualize microbes in nematodes from the wild or to experimentally characterize and quantify infection in nematodes infected with microbes in the lab. FISH probes, labeling the highly abundant small subunit ribosomal RNA, produce a bright signal for bacteria and microsporidian cells. Probes designed to target conserved regions of ribosomal RNA common to many species can detect a broad range of microbes, whereas targeting divergent regions of the ribosomal RNA is useful for narrower detection. Similarly, probes can be designed to label viral RNA. A protocol for RNA FISH staining with either paraformaldehyde (PFA) or acetone fixation is presented. PFA fixation is ideal for nematodes associated with bacteria, microsporidia, and viruses, whereas acetone fixation is necessary for the visualization of microsporida spores. Animals were first washed and fixed in paraformaldehyde or acetone. After fixation, FISH probes were incubated with samples to allow for the hybridization of probes to the desired target. The animals were again washed and then examined on microscope slides or using automated approaches. Overall, this FISH protocol enables detection, identification, and quantification of the microbes that inhabit the C. elegans intestine, including microbes for which there are no genetic tools available.

RNA Fluorescence in situ Hybridization FISH to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

Intestinal microbes, including extracellular bacteria and intracellular pathogens like the Orsay virus and microsporidia (fungi), are often associated with wild Caenorhabditis nematodes. This article presents a protocol for detecting and quantifying microbes that colonize and/or infect C. elegans nematodes, and for measuring pathogen load after controlled infections in the lab.

RNA蛍光 in situ ハイブリダイゼーション(FISH)による カエノラブディティスエレガンス 腸における微生物のコロニー形成と感染を可視化

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as ...

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.

Visualization of Bacteria in Bladder Biopsy Sections via Fluorescence In Situ Hybridization

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Visualization of Bacteria in Bladder Biopsy Sections via Fluorescence In Situ Hybridization