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.

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

12.2K Views.  Norfolk Academy. Het protocol is nuttig voor het verkrijgen van kwantitatief inzicht in de temporele regulatie van virale genproducten, binnen de context van gastheercellen, vooral met betrekking tot herpesvirussen. Deze techniek kan helpen bij het beantwoorden van onderzoeksvragen met betrekking tot zowel gastheer als virale RNAs en eiwitten. Bovendien is deze techniek compatibel met gelijktijdige biochemische testen.Om de cellen aan acht kamerdia's te hechten, breng 200 microliter van een inherente c...

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

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative tumor of vascular endothelial cells and produces rare B cell lymphoproliferative diseases in the form of pleural effusion lymphomas (PEL) and some forms of multicentric Castleman's disease.3-5 Only 1-5% of cells in KS lesions actively support lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent associated with KS, and it is clear that cellular factors must interact with viral factors in the process of oncogenesis and tumor progression.6,7 Identifying novel host-factor determinants which contribute to KS pathology is essential for developing prognostic markers for tumor progression and metastasis as well as for developing novel therapeutics for the treatment of KS.8 The accompanying video details the methods we use to identify host cell gene expression programs altered in dermal microvascular endothelial cells (DMVEC) after KSHV infection and in KS tumor tissue.9 Once dysregulated genes are identified by microarray analysis, changes in protein expression are confirmed by immunoblot and dual labeled immunofluorescence. Changes in transcriptional expression of dysregulated genes are confirmed in vitro by quantitative real-time polymerase chain reaction (qRT-PCR). Validation of in vitro findings using archival KS tumor tissue is also performed by dual labeled immunochemistry and tissue microarrays.8,10 Our approach to identifying dysregulated genes in the KS tumor tissue microenvironment will allow the development of in vitro and subsequently in vivo model systems for discovery and evaluation of potential novel therapeutic for the treatment of KS.

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus KSHV

Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative ...

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in determining outcomes that range from clearance to chronic persistent infections. We describe here a method for assessing the spatial and quantitative relationships between immune effector (E) virus-specific CD8+ T cells and infected targets (T) that combines in situ tetramer (IST) staining to detect virus-specific CD8+ T cells and in situ hybridization (ISH) to detect viral-RNA+ cells in the tissues where the battle between immune defenses and infection takes place. The combination of IST staining and ISH, abbreviated ISTH, enables visualization and mapping of the locations of immune effector cells and targets, and facile determination of E:T ratios.  These parameters in turn can then be used to determine the relationships between spatial proximity, and the timing and magnitude of the immune response that predict outcomes in early infection.

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

A technique combining in situ tetramer staining and in situ hybridization (ISTH) enables visualization, mapping and analysis of the spatial proximity of virus-specific CD8+ T cells to their virus-infected targets, and determination of the quantitative relationships between these immune effectors and targets to infection outcomes.

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in ...

Biology

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

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

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

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

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

Neuroscience

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 ...

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies 1-3. These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres 4-8.
Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell.
Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest 9. The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed.
This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase10. The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes11. In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.

A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and develops most often in the setting of immunosuppression. Despite decades of research, optimal treatment of KS remains poorly defined and clinical outcomes are especially unfavorable in resource-limited settings. KS lesions are driven by pathological angiogenesis, chronic inflammation, and oncogenesis, and various in vitro cell culture models have been developed to study these processes. KS arises from KSHV-infected cells of endothelial origin, so EC-lineage cells provide the most appropriate in vitro surrogates of the spindle cell precursor. However, because EC have a limited in vitro lifespan, and as the oncogenic mechanisms employed by KSHV are less efficient than those of other tumorigenic viruses, it has been difficult to assess the processes of transformation in primary or telomerase-immortalized EC. Therefore, a novel EC-based culture model was developed that readily supports transformation following infection with KSHV. Ectopic expression of the E6 and E7 genes of human papillomavirus type 16 allows for extended culture of age- and passage-matched mock- and KSHV-infected EC and supports the development of a truly transformed (i.e., tumorigenic) phenotype in infected cell cultures. This tractable and highly reproducible model of KS has facilitated the discovery of several essential signaling pathways with high potential for translation into clinical settings.

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is a tumor induced by infection with the oncogenic virus human herpesvirus-8/KS herpesvirus (HHV-8/KSHV). The endothelial cell culture model described here is uniquely suited for studying the mechanisms by which KSHV transforms host cells.

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and ...

Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to understand where and when transcription is taking place within nuclear space and to visualize the relationship between episomes infected within the same cell's nucleus. Here, both immunofluorescence (IFA) and RNA-FISH have been combinedto identify actively transcribing Kaposi's sarcoma-associated herpesvirus (KSHV) episomes. By staining KSHV latency-associated nuclear antigen (LANA), it is possible to locate where viral episomes exist within the nucleus. In addition, by designing RNA-FISH probes to target the intron region of a viral gene, which is expressed only during productive infection, nascent RNA transcripts can be located. Using this combination of molecular probes, it is possible to visualize the assembly of large viral transcription factories and analyze the spatial regulation of viral gene expression during KSHV reactivation. By including anti-RNA polymerase II antibody staining, one can also visualize the association between RNA polymerase II (RNAPII) aggregation and KSHV transcription during reactivation.

Viral transcriptional factories are discrete structures that are enriched with cellular RNA polymerase II to increase viral gene transcription during reactivation. Here, a method to locate sites of actively transcribing viral chromatin in 3D nuclear space by a combination of immunofluorescence staining and in situ RNA hybridization is described.

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to ...

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA fluorescence in situ hybridization (FISH) methods are often not compatible with immunostaining. We have therefore developed an imaging approach, MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and protein), which enables simultaneous single-cell visualization of DNA, RNA, and protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) ISH technology, which dramatically improves oligonucleotide probe sensitivity and detection. Small modifications of MICDDRP enable imaging of viral proteins concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have applied these protocols to study the life cycles of multiple viral pathogens, including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and influenza A virus (IAV). We demonstrated that we can efficiently label viral nucleic acids and proteins across a diverse range of viruses. These studies can provide us with improved mechanistic understanding of multiple viral systems, and in addition, serve as a template for application of multiplexed fluorescence imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.

Presented here is a protocol for a fluorescence imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and protein (MICDDRP), a method capable of simultaneous fluorescence single-cell visualization of viral protein and nucleic acids of different type and strandedness. This approach can be applied to a diverse range of systems.

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that ...

HCR-DNA FISH: A Fluorescence In Situ Hybridization Technique to Detect Viral DNA in Infected Cells

Source: Wei Liu&#160;et al., <a href="https://www.jove.com/v/58950"> Merkel Cell Polyomavirus Infection and Detection</a>, J. Vis. Exp. (2019).
In this video, the cells infected with MCPyV virions were subjected to in situ hybridization chain reaction to detect MCPyV specific genomes. Further, the DNA-HCR technique was combined with FISH to visualize the MCPyV DNA in infected human skin cells.

Source: Wei Liu&#160;et al.,  Merkel Cell Polyomavirus Infection and Detection, J. Vis. Exp. (2019).
In this video, the cells infected with MCPyV virions ...

JoVE Encyclopedia of Experiments

Cancer Research

Skin Cancer

Assays and techniques

Using Immuno-RNA Fluorescence In Situ Hybridization to Visualize SARS-CoV-2

Source: Kula-Pacurar, A., et al. <a href="https://app.jove.com/v/62067">Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization.</a> J. Vis. Exp. (166), (2020).
This video demonstrates the application of Hybridization Chain Reaction (HCR)-Fluorescence In Situ Hybridization (FISH) for visualizing the spatial distribution of SARS-CoV-2 subgenomic RNA. Combining HCR with immunofluorescence techniques has the potential to provide insights into host-virus interactions at the single-cell level.

1. SARS-CoV-2 RNA-FISH in Vero cells cultured on coverslips
DAY 1 

	Fixing and permeabilizing cells&#8203;
	
		Fix infected cells with 3.7% w/v PFA ...

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

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