We thank Jonathan Rodenfels, Kazimierz Tycowski, and Johanna B. Withers for advice on data analysis. We also thank G. Hayward for the anti-SSB antibody. This work was supported by grants T32GM007223 and T32AI055403 from the National Institutes of Health (to TKV) and NIH grant (CA16038) (to JAS). JAS is an investigator of the Howard Hughes Medical Institute. Figures 1-3&#160;and Table 1 were reproduced with permission from the American Society for Microbiology under a Creative Commons Attribution license from the following publication: Vallery, T. K., Withers, J. B., Andoh, J. A., Steitz, J. A. Kaposi&#39;s Sarcoma-Associated Herpesvirus mRNA Accumulation in Nuclear Foci Is Influenced by Viral DNA Replication and Viral Noncoding Polyadenylated Nuclear RNA. Journal of Virology.&#160;92 (13), doi:10.1128/JVI.00220-18, (2018).

1. Design of fluorescence in situ (FISH) anti-sense oligonucleotides to detect a specific herpesviral transcript
<ol>
	<li>Select 25 to 40 nt segments from the sequence of RNA of interest and convert to be anti-sense. A successful FISH strategy may contain from one up to ten or more different anti-sense oligonucleotides. When selecting sequences, consider the following:
	<ol>
		<li>If the RNA of interest contains a unique repeat region, then capitalize on this feature and design an anti-sense oligonucleotide to target the repeat sequence. 
		NOTE: Tycowski and colleagues5 provide an example of this strategy with rhesus rhadinovirus (RRV) polyadenylated nuclear (PAN) RNA.</li>
		<li>If the RNA of interest contains a known protein-binding site or stem-loop structure, design oligonucleotides that avoid these regions.</li>
		<li>Depending on the goals of the experiments, consider the intronic sequence and whether or not to design an anti-sense oligonucleotide for it.</li>
	</ol>
	</li>
	<li>Perform simple computational analyses on the selected anti-sense sequences to ensure binding specificity and reduce aggregation of the anti-sense oligonucleotide.
	<ol>
		<li>The sequences must be approximately 50% GC-rich (high guanine and cytosine content) and have a melting temperature in the range of 60 to 70 &#176;C.</li>
		<li>Use a sequence analyzer tool to select sequences that do not self-dimerize or form hairpins with melting temperatures above 37 &#176;C, the hybridization temperature.</li>
		<li>Perform a NCBI BLASTn (National Center for Biotechnology Information Basic Local Alignment Search Tool for nucleotide alignments) search of the selected sequences against both the host and viral transcriptomes using the &#39;somewhat similar&#39; setting. This search will identify unique anti-sense oligonucleotides that will not likely bind to other host or viral transcripts. 
		NOTE: If transcriptomes are not available, perform the BLAST search with the genomic sequences. It is ideal if the searches are performed on the sequences from the virus isolated from the infected cells used in the experiment because wild strains tend to diversify and contain a combination of sequences from different lab strains.</li>
	</ol>
	</li>
	<li>Order purified DNA oligonucleotides corresponding to the anti-sense sequence that has been verified computationally to be unique and likely to bind to the target RNA. No special modifications need to be introduced into the oligonucleotides.</li>
	<li>Test the designed anti-sense oligonucleotides for binding specificity by FISH and Northern blot.
	<ol>
		<li>Using an uninfected cell-line from the same host species (e.g., 293T) and ideally the same cell type, conduct a transfection with a plasmid expressing the RNA of interest from a robust promoter (CMV, cytomegalovirus) and one with the empty vector (e.g., pcDNA3). Use a positive control for transfection such as co-transfection with a GFP (green fluorescent protein) plasmid (e.g., pmaxGFP) or the vector containing GFP. 
		NOTE: It is important to avoid cell-lines that have been immortalized using the herpesvirus Epstein-Barr virus (EBV) since there are sequence similarities between herpesviruses.</li>
		<li>Perform FISH as described in section 3 on both sets of cells with the anti-sense oligonucleotides as described in this protocol. Deduce the successful candidates by comparing FISH experiments with individual, pairs, or sets of the anti-sense oligonucleotides. Use a positive control for the FISH protocol such U2 snRNA (small nuclear RNA) FISH, present at 500,000 copies per human cell nucleus6 (Table 1).</li>
		<li>The fluorescent signal should be specific and strong in the cell containing the RNA of interest. Design additional anti-sense oligonucleotides to strengthen the signal and remove anti-sense oligonucleotides that bind nonspecifically from consideration. Signal strength must be above background and autofluorescence.</li>
		<li>Test binding specificity by Northern blot.</li>
	</ol>
	</li>
</ol>
2. Oligonucleotide and cell preparation
<ol>
	<li>Following the manufacturer&#39;s instructions, use terminal transferase to label the anti-sense oligonucleotides with dioxigenin(DIG)-dUTP or, if strongly binding, directly with a fluorescent nucleotide like Alexa Fluor 594-5-dUTP. After labeling, additional purification is not necessary. Store labeled oligonucleotides at -20 &#176;C up to several years and in tin foil if directly labeled to prevent photobleaching. 
	CAUTION: The labeling solution contains a toxic material, potassium cacodylate. Handle labeling reactions with gloves. 
	NOTE: To conserve resources, several different anti-sense oligonucleotides can be labeled in one reaction. This protocol utilizes 3&#39;-end labeling. Internal labeling is challenging because the chemical group (e.g., DIG or the fluorophore) gets caught or is unable&#160;to enter the active site of a DNA polymerase. An experiment with two different RNAs can be performed using directly-labeled anti-sense oligonucleotides and anti-DIG immunofluorescence with a different fluorophore (e.g., FITC (fluorescein) or Alexa Fluor 488 with Alexa Fluor 594).</li>
	<li>Adhere the cells to the eight-chamber slides. 
	NOTE: Eight-chamber slides allow several simultaneous experiments while minimizing precious resources like antibodies. An alternative to eight-chamber slides is a six-well tissue culture plate with standard coverslips (22 mm x 22 mm) that are both sterile. A similar arrangement is possible with circular coverslips and a 24-well tissue culture plate. For both, increase the volumes mentioned in this protocol by 10-15x (e.g., 1.75 mL hybridization solution), and 4x (e.g., 600 &#181;L hybridization solution) respectively.
	<ol>
		<li>For adherent lytic cells, use 1x trypsin/PBS at 37 &#176;C and 5% CO2 for 10 min to suspend cells and dilute to 60% confluency. 
		NOTE: Adherent cell lines used in FISH experiments included 293T, iSLK.2197, and iSLK-BAC36 cells8.</li>
		<li>Apply 200 &#181;L of cell suspension to each chamber of the sterile eight-chambered slides and allow seed growth for 12-24 h at 37 &#176;C and 5% CO2. Adjust as necessary for slow- or fast-growing cells and for cells that are easily damaged by trypsin. 
		NOTE: The objective is to have evenly spaced cells firmly attached to the slide. Consider inducing lytic phase after adhesion if the lytic cells are fragile. Conclusions drawn from experiments with iSLK cells are limited9.</li>
		<li>For lytic suspension-cells, pre-treat eight-chamber slides with 1:10 poly L-lysine for 5 min under the tissue culture hood. Then leave the slides to dry overnight at room temperature or 1 h at 65 &#176;C. Incubate 800 &#181;L of lytic cells at a concentration of 1 x 106 cells/mL with the chambered slides for 30 min to 1 h at 37 &#176;C and 5% CO2. 
		NOTE: Suspension-cells will settle in a monolayer, sticking to the poly L-lysine and thus excess cells are not a concern in comparison to adherent cells. Lytic cells that form grape clusters, if possible, should be separated by gentle vortexing or chemical means. Admittedly, the authors have not had much success with such recommendations in the case of lytic BJAB-RRV-GFP cells. If suspension-cells do not adhere well, consider increasing either the time or concentration of the poly L-lysine incubation.</li>
	</ol>
	</li>
</ol>
3. Fixation, Immunofluorescence (Optional), Hybridization, and Visualization of Viral RNAs
<ol>
	<li>Remove media and excess cells. Throughout this protocol, use vacuum suction to remove solutions and gentle micropipetting to add solutions. 
	NOTE: The strength of a vacuum can be reduced by placing a 200 &#181;L micropipette tip over the glass Pasteur pipette. Replace the micropipette tip between wash steps to prevent contamination. Each wash step must be performed quickly because it is imperative that the cells never dry out.</li>
	<li>Immediately, fix the cells with pre-chilled 4% formaldehyde/PBS (phosphate-buffered saline) on ice for 30 min. Wash the cells three times with 200 &#181;L 1x PBS cooled to 4 &#176;C and incubate for 5 min at room temperature or on ice.
	<ol>
		<li>Permeabilize the fixed cells with 200 &#181;L of pre-chilled 0.5% Triton-X/PBS (phosphate buffered saline) for 10 min on ice or 750 &#181;L of pre-chilled 70% ethanol at 4 &#176;C for 1 h (min) to 7 d (max). 
		NOTE: Collect protein, total RNA, and genomic DNA samples at the point of fixation to ensure consistency between images and biochemical assays. All washes throughout this protocol are performed in the same manner unless otherwise specified. 70% ethanol loosens the glue between the chambers and the slide, which eases later separation, and also provides a significant pause in the protocol. Nonetheless, use paraffin film around the chamber slide to reduce evaporation and check the level of the ethanol in each chamber about every 8 h. 70% ethanol also flattens the cells, making a crisper image, while Triton-X does not dehydrate the cells and change the dimensions of the cell.</li>
	</ol>
	</li>
	<li>Remove chambers carefully to prevent cracking the slide. If the experiment includes immunofluorescence (IF) of a viral or host protein with a polyclonal primary antibody, perform the IF as described below before proceeding to RNA FISH. If the immunofluorescence uses a monoclonal primary antibody, then perform immunofluorescence as described in step 3.3.1 after step 3.11. 
	NOTE: Use a fresh removal device or one with very little leftover adhesive provided by the&#160;manufacturer and gently ease the chambers off to prevent the slide from cracking. Using 70% ethanol as the permeabilizing reagent for 4 h greatly reduces the likelihood of cracking. In the case of a crack, continue the protocol on chambers not affected by the crack and be mindful of the higher oxidation&#160;rate of imperfectly sealed slides (i.e. decreased storage&#160;life).
	<ol>
		<li>Rinse cells with pre-chilled 1x PBS and block with pre-chilled 4% BSA (bovine serum albumin)/1x PBS for 30 min at 4 &#176;C. 
		NOTE: The use of BSA throughout this protocol limits nonspecific labeling.</li>
		<li>Remove blocking solution and incubate the cells with 1:200 or another polyclonal primary antibody in 0.1% BSA/1x PBS for 1 h at 4 &#176;C. Then wash three times with 1x PBS. 
		NOTE: An antibody10 for detection of SSB/ORF6 (viral single-stranded DNA binding protein) was used at 1:200 dilution.</li>
		<li>Incubate the cells with a secondary antibody with fluorophore compatible with the FISH-detecting antibody for 1 h at 4 &#176;C. Wash three times with 1x PBS. Then fix with 4% formaldehyde/1x PBS for 10-15 min and permeabilize with either Triton-X or 70% ethanol as previously described before proceeding to FISH. Cover slide with tin foil to preserve fluorescent signal and prevent photobleaching.</li>
	</ol>
	</li>
	<li>Wash the cells with 2x SSC (saline sodium citrate) once and then apply 45 &#181;L of hybridization solution consisting of 50% formamide, 10% dextran sulfate, 2x SSC, 0.1% BSA, 500 &#181;g/mL salmon sperm DNA, 125 &#181;g/mL E. coli tRNA, and 1 mM vanadyl ribonucleoside complexes. Incubate for 1 h at 37 &#176;C in a humidity chamber that can be a 150 mm Petri dish with moistened sterile wipes. 
	NOTE: Prepare fresh hybridization solution at least an hour before use. Dissolve the dextran sulfate in water first, vortexing frequently and incubating in a 37 &#176;C water bath.</li>
	<li>Calculate to have a suggested concentration of 25 &#181;M oligonucleotides in 35-uL hybridization solution per chamber. Adjust concentration of anti-sense oligonucleotide as needed. Add distilled water to the oligonucleotides to bring the denaturation volume to 10 &#181;L. 
	NOTE: Following the labeling reaction, the oligonucleotides are stored in the quenched solution containing 0.18 M potassium cacodylate, 23 mM Tris-HCl, 0.23 mg/mL BSA, 4.5 mM CoCl2, 18 mM EDTA, 2.7 mM K-phosphate, and 6.8 mM KCl, 45 &#181;M 2-Mercaptoethanol, 0.02% Triton X-100, and 2% glycerol. The concentrations are high enough that dilution with water will bring the denaturation solution to concentrations near to 1x TE (10 mM Tris-HCl and 1 mM EDTA), a standard oligonucleotide denaturation buffer.</li>
	<li>Denature the DIG- and/or Alexa Fluor 594-labeled oligonucleotides at 95 &#176;C for 5 min. Then add 35 &#181;L fresh hybridization solution per intended chamber to the denatured oligonucleotides. If performing double FISH, both sets of anti-sense oligonucleotides may be denatured and hybridized together.</li>
	<li>Remove the pre-hybridization solution and then add hybridization solution containing the labeled oligonucleotides to the cells. Incubate overnight in the humidity chamber at 37 &#176;C with tin foil to protect the fluorophore-labeled oligonucleotides. 
	NOTE: Incubation should be at least 10 h and not more than 24 h.</li>
	<li>The next day, wash the cells twice with 2x SSC for 10 min at 37 &#176;C and then twice with 1x SSC for 10 min at 25 &#176;C.</li>
	<li>Fix the cells with pre-chilled 4% formaldehyde/1x PBS for 10-15 min on ice. Then wash the cells with PBS three times and permeabilize for 1 h with pre-chilled 70% ethanol or for 10 min with pre-chilled 0.5% Triton-X/1x PBS at 4 &#176;C.</li>
	<li>Incubate the cells with 1:200 anti-DIG FITC in pre-chilled 0.1% BSA/1x PBS for 1 h at 4 &#176;C. Remove the antibody solution and wash three times with 1x PBS.</li>
	<li>Fix with pre-chilled 4% formaldehyde/1x PBS for 10-15 min at 4 &#176;C and then wash three times with 1x PBS. If performing immunofluorescence for a host or viral protein with a monoclonal primary antibody, permeabilize the cells and then perform the IF protocol outlined in step 3.3.1. Otherwise proceed to the DAPI staining.</li>
	<li>Incubate the cells with 0.4 &#181;g/mL DAPI in pre-chilled 0.5% Triton-X/1x PBS for 15 min on ice and then wash three times with 1x PBS.</li>
	<li>Mount slides with fluorescent beads (optional) and a mounting medium. Then seal the coverslip to the slide with clear nail polish.</li>
	<li>Using a confocal microscope, collect images of the samples within an hour to a week of performing the protocol at 630x magnification. Apply multiple coats of nail polish to seal the cover slip and to prolong fluorophore life by reducing the rate of oxidation. 
	NOTE: Do not use a DAPI-containing mounting medium. When collecting the images, include the scale bar on each image for later quantification. Fluorescent beads serve as controls of fluorescence intensity between slides and sample preparations11. Acquire images at the midsection of the cell for two-dimensional (2D) quantification in step 4.</li>
</ol>
4. Quantification of FISH and IF images to highlight subcellular localization and to determine nucleocytoplasmic ratio of fluorescence
<ol>
	<li>Perform image analysis on an assembled stack of the various fluorescent-stained and merged images to ensure consistency. Set the scale of the image analysis software using the scale bar included when the images were collected.</li>
	<li>To quantify fluorescence intensity across several channels and in reference to the nuclear DAPI stain, use a line tool and a plot-profile function. Then indicate the line permanently on a copy of the image using markers that do not obstruct or influence the viewer&#39;s judgment.
	<ol>
		<li>Establish criteria to guide where the line is drawn such as a trace that captures a diversity of topographical features, peaks and valleys, along a central axis or a line that does not traverse supersaturated areas. 
		NOTE: These line traces depict raw fluorescence in a cell and thus are limited to comparisons of the locations of a stain, not intensity. To compare intensities of the same stain between slides, treatments, or preparations, add a fluorescent bead to the slide as an internal control during step 3.13. The fluorescent bead must be added during the mounting process and detected with the same settings on the excitation laser and photomultiplier tube (confocal).</li>
	</ol>
	</li>
	<li>To quantify a shift in subcellular localization, calculate nucleocytoplasmic ratios of cells undergoing different treatments.
	<ol>
		<li>Measure the area and raw fluorescence intensity of both the nucleus and cytoplasm using the nuclear DAPI stain to set the inner boundary. Include nuclear and cytoplasmic controls such as a nuclear RNA (e.g., KSHV PAN RNA) and cytoplasmic RNA (e.g., host GAPDH mRNA). Moreover, calculate background intensity for three cell-like areas and average the values per pixel or &#181;m2. 
		NOTE: Intensity values tend to lack units and so the term &#39;units&#39; is used.</li>
		<li>Normalize both nuclear and cellular raw intensity values by first determining the average background for the same area and then subtracting that individualized value from the raw intensity of the area.
		<ol>
			<li>For example, a nucleus of a lytic B-cell has an area of 133.4 &#181;m2 and a raw intensity of 75976 units while the background intensity for the same fluorescent signal was determined to be 0.67 units per &#181;m2. The normalized nuclear intensity would be 
			<img alt="Equation 1" src="/files/ftp_upload/59697/59697eq1.jpg" style="opacity: 0.9;" /></li>
		</ol>
		</li>
		<li>Enter the values in the following equation. 
		<img alt="Equation 2" src="/files/ftp_upload/59697/59697eq2.jpg" style="opacity: 0.9;" /> 
		NOTE: This calculation controls for changes in subcellular area. Lytic induction and drug treatments can enlarge the nucleus or change the size of the cell, respectively.</li>
		<li>To interpret the results, create a box whisker plot. An equal distribution of the fluorescent signal would be close to zero, whereas a nuclear distribution would favor a positive ratio value and a cytoplasmic distribution would trend toward a negative ratio value.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interest to disclose.

The protocol described in this report can be adapted to different cell types and includes steps for double RNA FISH and RNA FISH with IF using both monoclonal and polyclonal primary antibodies. Although prepared slides are typically imaged with a confocal microscope, imaging can be performed with a STED (stimulated emission depletion) microscope after modifications of increased antibody concentration and a different mounting medium. For enhanced analysis of individual cells, samples prepared with this protocol may also b...

In their lytic (active) phase, herpesviruses hijack the host cell, causing changes in cell morphology and localization of biological molecules, to produce virions. The base of operations is the nucleus, where the double-stranded DNA viral genome is replicated and packaged into a protein shell, called a capsid1. To begin, the virus expresses its own proteins, hijacking host machinery and preventing expression of non-essential host genes, a process termed the host shutoff effect. The majority of this activity is localized to specific 4&#8242;,6-diamidino-2-phenylindole (DAPI)-free nuclear regions called viral replication compartments, comprised of both host and viral proteins, RNAs, and viral DNA2. The cell is overhauled to provide space and resources for the replication compartments and thus assembly of viral capsids. Once the capsid exits the nucleus, how the capsid is enveloped in the cytoplasm to produce a membrane-bound viral particle, also known as a virion, is unclear. Understanding of the localization and spatial shifts of both host and viral biomolecules during the lytic phase provides deeper mechanistic insight into the arrangement of the replication compartment, host shutoff effect, the virion-egress pathway, and other processes related to herpesviral infection and replication.
Currently the best method to detect and study these changes is the visualization of proteins and RNAs in infected cells with immunofluorescence (IF) and fluorescent in situ hybridization (FISH), respectively. Use of a time-course with these techniques reveals the localization of biomolecules at key points of the lytic phase or simply, spatiotemporal data. FISH and IF complement other biochemical techniques, such as inhibition of a cellular process (e.g., inhibition of viral DNA replication), RT-qPCR (real-time polymerase chain reaction), RNA sequencing, Northern blots, mass spectrometry, Western blotting, and analysis of viral DNA production, that may provide a more global picture of cellular activities.
We developed RNA FISH strategies to examine the RNA products from specific genes and a computational analysis that quantitatively calculates the nucleocytoplasmic ratio of a specific gene product. The sample preparation, modified from earlier publications by Steitz and colleagues3,4, is relatively easy and can be used for both adherent and suspended cells. The protocol is also adaptable for simultaneous use of multiple RNA FISH strategies (double RNA FISH) or RNA FISH with IF strategies. Development of a specific FISH strategy is challenging, but suggestions to improve success are outlined. The data analysis described here is quantitative if fluorescent beads and strong markers of compartment boundaries are used and offers additional insight into the micrographs, insight that removes observation bias. The detailed protocol is designed for both latent and lytic cells infected by Kaposi&#39;s sarcoma-associated herpesvirus (KSHV) and can be used with uninfected cells or cells infected by other herpesviruses5. The methods of quantitation are applicable to studies on nucleocytoplasmic shifts or relocalization between subcellular compartments in most cells.

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			<td height="21" style="height:21px;width:361px;">AlexaFluor594-5-dUTP</td>
			<td style="width:191px;">Life Technologies</td>
			<td style="width:117px;">C1100</td>
			<td style="width:277px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-DIG FITC&#160;</td>
			<td>Jackson Lab Immunologicals</td>
			<td>200-092-156</td>
			<td style="width:277px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Rabbit Secondary AlexaFluor594 Monoclonal Antibody</td>
			<td>Invitrogen</td>
			<td>A-11037</td>
			<td>Goat</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-SSB Antibody</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Ref. Chiou et al. 2002</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BLASTn</td>
			<td>NIH NCBI</td>
			<td>N/A</td>
			<td style="width:277px;">Free Sequence Alignment Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextran Sulfate</td>
			<td>Sigma Aldrich</td>
			<td>D8906</td>
			<td style="width:277px;">Molecular Biology Grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DIG-Oligonucleotide Tailing Kit</td>
			<td>Sigma Roche</td>
			<td>#03353583910</td>
			<td style="width:277px;">2nd Gen</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Eight-Chamber Slides</td>
			<td>Nunc Lab Tek II</td>
			<td>#154453</td>
			<td style="width:277px;">Blue seal promotes surface tension but separation by clear gel is also available.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formamide</td>
			<td>Sigma Aldrich</td>
			<td>F9037</td>
			<td style="width:277px;">Molecular Biology Grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GAPDH Probes</td>
			<td>Stellaris</td>
			<td>SMF-2019-1</td>
			<td style="width:277px;">Compatible with protocol, Quasar 670</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">ImageJ&#160;</td>
			<td>NIH, Bethesda, MD</td>
			<td>N/A</td>
			<td style="width:277px;">Free Image Analysis Software, [http:rsb.info.nih.gov/ij/]</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OligoAnalyzer</td>
			<td>IDT</td>
			<td>N/A</td>
			<td style="width:277px;">Free Oligonucleotide Analyzer&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pcDNA3</td>
			<td>Invitrogen</td>
			<td>A-150228</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pmaxGFP</td>
			<td>Amaxa</td>
			<td>VDF-1012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly L-Lysine</td>
			<td>Sigma Aldrich</td>
			<td>P8920</td>
			<td style="width:277px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Terminal Transferase</td>
			<td>Sigma Roche</td>
			<td>#003333574001</td>
			<td style="width:277px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vanadyl Ribonucleoside Complexes&#160;</td>
			<td>NEB</td>
			<td>S1402S</td>
			<td style="width:277px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Vectashield</td>
			<td>Vector Laboratories, Inc.&#160;</td>
			<td>H-1000</td>
			<td style="width:277px;">DAPI within the mounting media scatters the light and reduces contrast.&#160;</td>
		</tr>
	</tbody>
</table>

quantitative fluorescence in situ hybridization (fish) and immunofluorescence (if) of specific gene products in kshv-infected cells

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout the cell at specific times can be captured with fluorescence in situ hybridization (FISH) and immunofluorescence (IF). During lytic herpesvirus infection, the virus hijacks the host cell to preferentially express viral genes, causing changes in cell morphology and behavior of biomolecules. Lytic activities are centered in nuclear factories, termed viral replication compartments, which are discernable only with FISH and IF. Here we describe an adaptable protocol of RNA FISH and IF techniques for Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells, both adherent and in suspension. The method includes steps for the development of specific anti-sense oligonucleotides, double RNA FISH, RNA FISH with IF, and quantitative calculations of fluorescence intensities. This protocol has been successfully applied to multiple cell types, uninfected cells, latent cells, lytic cells, time-courses, and cells treated with inhibitors to analyze the spatiotemporal activities of specific RNAs and proteins from both the human host and KSHV.

The FISH and IF methods detailed in this manuscript are shown in Figure 1 along with the quantification of results by line traces of fluorescent intensity. The results presented here are semi-quantitative and offer insight into localization, rather than into comparisons between intensities of different fluorescent stains because experiments did not include a fluorescent bead in the slide preparation. Figure 1 also reveals that th...

Watch this Scientific Journal Video about Quantitative Fluorescence In Situ Hybridization FISH and Immunofluorescence IF of Specific Gene Products in KSHV-Infected Cells at JoVE.com

Quantitative Fluorescence In Situ Hybridization FISH and Immunofluorescence IF of Specific Gene Products in KSHV-Infected Cells

We describe a protocol utilizing fluorescence in situ hybridization (FISH) to visualize multiple herpesviral RNAs within lytically infected human cells, either in suspension or adherent. This protocol includes quantification of fluorescence producing a nucleocytoplasmic ratio and can be extended for simultaneous visualization of host and viral proteins with immunofluorescence (IF).

Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF) of Specific Gene Products in KSHV-Infected Cells

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout ...

quantitative-fluorescence-situ-hybridization-fish-immunofluorescence

Research

JoVE Journal

Immunology and Infection

12.0K Views.  Norfolk Academy. We describe a protocol utilizing fluorescence in situ hybridization (FISH) to visualize multiple herpesviral RNAs within lytically infected human cells, either in suspension or adherent. This protocol includes quantification of fluorescence producing a nucleocytoplasmic ratio and can be extended for simultaneous visualization of host and viral proteins with immunofluorescence (IF). 

Video: Quantitative Fluorescence In Situ Hybridization FISH and Immunofluorescence IF of Specific Gene Products in KSHV-Infected Cells

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.
Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.
Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes. Investigation of transcripts and gene regulators that are expressed in tissues in which the mosquito host and pathogen interact, and in organs involved in reproduction are of great interest for strategies to reduce mosquito-borne disease transmission and disrupt egg development. A number of tools have been employed to study and validate the temporal and tissue-specific regulation of gene expression. Here, we describe protocols that have been developed to obtain spatial information, which enhances our understanding of where specific genes are expressed and their products accumulate. The protocol described has been used to validate expression and determine accumulation patterns of transcripts in tissues related to mosquito-borne pathogen transmission, such as female salivary glands, as well as subcellular compartments of ovaries and embryos, which relate to mosquito reproduction and development. 
 The following procedures represent an optimized methodology that improves the efficiency of various steps in the protocol without loss of target-specific hybridization signals. Guidelines for RNA probe preparation, dissection of soft tissues and the general procedure for fixation and hybridization are described in Part A, while steps specific for the collection, fixation, pre-hybridization and hybridization of mosquito embryos are detailed in Part B.

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes.  Investigation of transcripts and gene ...

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Fluorescence in situ Hybridizations (FISH) for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells and can be further modified to visualize other components in the cell such as infection with viruses and bacteria. Spatial localization and visualization of viruses and bacteria during the infection process is an essential step that complements expression profiling experiments such as microarrays and RNAseq in response to different stimuli. Understanding the spatiotemporal infections with these agents complements biological experiments aimed at understanding their interaction with cellular components. Several techniques for visualizing viruses and bacteria such as reporter gene systems or immunohistochemical methods are time-consuming, and some are limited to work with model organisms&#160;and involve complex methodologies. FISH that targets RNA or DNA species in the cell is a relatively easy and fast method for studying spatiotemporal localization of genes and for diagnostic purposes. This method can be robust and relatively easy to implement when the protocols employ short hybridizing, commercially-purchased probes, which are not expensive. This is particularly robust when sample preparation, fixation, hybridization, and microscopic visualization do not involve complex steps. Here we describe a protocol for localization of bacteria and viruses in insect and plant tissues. The method is based on simple preparation, fixation, and hybridization of insect whole mounts and dissected organs or hand-made plant sections, with 20 base pairs short DNA probes conjugated to fluorescent dyes on their 5&#39; or 3&#39; ends. This protocol has been successfully applied to a number of insect and plant tissues, and can be used to analyze expression of mRNAs or other RNA or DNA species in the cell.

Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

We describe here a simple fluorescence in situ hybridization (FISH) method for the localization of viruses and bacteria in insect and plant tissues. This protocol can be extended for the visualization of mRNA in whole mount and microscopic sections.

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells ...

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in B-cell and plasma-cell production of antibodies, and detecting and eliminating cancer cells. The development of MHC-tetramer staining of antigen-specific T cells analyzed by flow cytometry has revolutionized our ability to study and understand the immunobiology of T cells. While extremely useful for determining the quantity and phenotype of antigen-specific T cells, flow cytometry cannot determine the spatial localization of antigen-specific T cells to other cells and structures in tissues, and current disaggregation techniques to extract the T cells needed for flow cytometry have limited effectiveness in non-lymphoid tissues. In situ MHC-tetramer staining (IST) is a technique to visualize T cells that are specific for antigens of interest in tissues. In combination with immunohistochemistry (IHC), IST can determine the abundance, location, and phenotype of antigen-specific CD8 and CD4 T cells in tissues. Here, we describe a protocol to stain and enumerate antigen-specific CD8 T cells, with specific phenotypes located within specific tissue compartments. These procedures are the same that we used in our recent publication by Li et al., entitled &#34;Simian Immunodeficiency Virus-Producing Cells in Follicles Are Partially Suppressed by CD8+ Cells In Vivo.&#34; The methods described are broadly applicable because they can be used to localize, phenotype, and quantify essentially any antigen-specific CD8 T cell for which MHC tetramers are available, in any tissue.

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

Here, we describe a method that combines in situ MHC-tetramer staining with immunohistochemistry to determine localization, phenotype, and quantity of antigen-specific T cells in tissues. This protocol is used to determine the spatial and phenotypic characteristics of antigen-specific CD8 T cells relative to other cell type and structures in tissues.

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...