This work was supported by the Ministry of Science and Higher Education for research on SARS-CoV-2, and by grants from the National Science Center (grants UMO2017/27/B/NZ6/02488 to K.P. and UMO-2018/30/E/NZ1/00874 to A.K.-P.).

1. Buffer preparation
<ol>
	<li>For 500 mL of 2x PHEM buffer, combine 18.14 g of piperazine-N,N&#8242;-bis(2-ethanesulfonic acid) (PIPES), 6.5 g of 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), 3.8 g of ethylene glycol tetraacetic acid (EGTA), and 0.99 g of magnesium sulfate (MgSO4). Make the volume up to ~400 mL with distilled water (dH2O), stir, and adjust the pH to 7.0 using 10 M potassium hydroxide (KOH) or sodium hydroxide (NaOH). Make the final volume up to 500 mL, and then split into 50 mL aliquots. Store at -20 &#176;C until required. 
	NOTE: The buffer will not be clear until the pH reaches 7.0.</li>
	<li>Prepare a stock solution of 37% w/v paraformaldehyde (PFA). For 50 mL, mix 18.5 g of PFA and 35 mL of dH2O in a glass bottle. Place the bottle on a magnetic stirrer with heating. Add 900 &#181;L of 1 M KOH or NaOH, and stir until the solution becomes clear. Allow to cool and transfer to a 50 mL conical centrifuge tube; top up to 50 mL with dH2O. The solution can be stored at -20 &#176;C until required. 
	NOTE: Formaldehyde should be handled in a fume hood while wearing protective gloves and a lab coat.</li>
	<li>Prepare fixation buffer (3.7% PFA buffered with PHEM). For 50 mL, combine 5 mL of 37% PFA solution, 25 mL of 2x PHEM buffer, and 20 mL of dH2O in a 50 mL conical centrifuge tube. Store at -20 &#176;C until required. 
	NOTE: After thawing, the buffer can be stored at 4 &#176;C for up to 3 months.</li>
	<li>Prepare PBST (0.1% Tween-20 in 1x phosphate-buffered saline [PBS]). For 50 mL, add 50 &#181;L of 100% Tween-20 to 50 mL of 1x PBS and mix well. 
	NOTE: The solution can be stored at room temperature (RT).</li>
	<li>Prepare rehydration buffers by combining MeOH/PBST in ratios of 3:1, 1:1, and 1:3 (final volume of 100 mL of each; see Table 1). 
	NOTE: MeOH is very toxic and flammable. As it can damage the optic nerve, it should be handled in a fume hood avoiding any open flames. As mixing MeOH and PBST generates an exothermic reaction, combine the solutions on ice.</li>
	<li>For 1000 mL of 20x SSC, combine 175.3 g of sodium chloride (NaCl) and 88.2 g of sodium citrate in a beaker, and fill up with 800 mL of dH2O. Stir until dissolved, and adjust the pH to 7.2 using NaOH. Add dH2O to a total volume of 1000 mL, and autoclave or filter through a 0.22 &#181;m filter into an autoclaved bottle.</li>
	<li>For 50 mL of 5x SSCT, combine 12.5 mL of 20x SSC buffer with 37.5 mL of dH2O, and add 50 &#181;L of 100% Tween-20. Mix well.</li>
	<li>For 50 mL of 50% 5x SSCT/50% PBST, combine 25 mL of 5x SSCT with 25 mL of PBST.</li>
	<li>For 50 mL of 2x SSC, combine 5 mL of 10x SSC buffer with 45 mL of dH2O. Mix well. 
	&#8203;NOTE: All SSC buffers should be stored at RT in the dark.</li>
</ol>
2. Target definition, probes, and amplifiers
<ol>
	<li>Use the tools available on the manufacturer&#39;s website to design amplifiers and probes. Ensure that the probes are complementary to the reverse DNA strand of the SARS-CoV-2 N gene (Supplementary Figure 1).</li>
	<li>Determine the best probe set size (a set of 20 probes is sufficient for visualization).</li>
	<li>Set the amplifiers used for target RNA detection.
	<ol>
		<li>Use HCR amplifier B1 labeled with Alexa Fluor 647. 
		NOTE: An HCR amplifier comprises metastable HCR hairpins h1 and h2. Multiplexed experiments can be designed using this protocol. If this is planned, choose a different HCR amplifier (B1, B2, &#8230;) for each target RNA to be imaged within the same sample (e.g., amplifier B1 for target 1, amplifier B2 for target 2, &#8230;).</li>
	</ol>
	</li>
</ol>
3. Cell culture and infection with SARS-CoV-2
<ol>
	<li>Culture Vero (monkey kidney epithelial cells) cells in Dulbecco&#39;s modified Eagle medium containing 5% fetal bovine serum.
	<ol>
		<li>Seed 50,000 cells onto coverslips (No. 1, 15 &#215; 15 mm) in a 12-well plate.</li>
	</ol>
	</li>
	<li>Prepare fully differentiated HAE cultures as described18 on permeable Transwell insert supports with a membrane (diameter = 6.5 mm) and culture in bronchial epithelial growth medium until fully confluent.</li>
	<li>Virus infection
	<ol>
		<li>Inoculate cells with SARS-CoV-2 at 1000x median tissue culture infectious dose (TCID50) per mL</li>
		<li>Incubate cells for 2 h at 37 &#176;C.</li>
		<li>Wash cells twice with PBS to remove unbound virus.</li>
		<li>Culture cells for 48 h.</li>
	</ol>
	</li>
</ol>
4. SARS-CoV-2 RNA-FISH in Vero cells cultured on coverslips
DAY 1
<ol>
	<li>Fixing and permeabilizing cells
	<ol>
		<li>Fix infected cells with 3.7% w/v PFA solution for 1 h at RT.</li>
		<li>Aspirate the 3.7% PFA solution, and wash the cells twice using 1x PBS.</li>
		<li>Permeabilize the cells with PBST solution for 10 min at RT with agitation.</li>
		<li>Aspirate the PBST, and wash the cells twice with 1x PBS.</li>
	</ol>
	</li>
	<li>Detection
	<ol>
		<li>Aspirate the 1x PBS solution, and wash the cells twice with 2x SSC at RT.</li>
		<li>Aspirate the 2x SSC solution, and prehybridize the samples by adding at least 300 &#181;L of probe hybridization buffer. Cover the wells containing the cells, and incubate at 37 &#176;C for 30 min.</li>
		<li>Prepare the probe solution by adding 1.2 pmol of probe mixture to the probe hybridization buffer.
		<ol>
			<li>Use 1.2 &#181;L of the 1 &#181;M probe stock to prepare 300 &#181;L of working stock.</li>
		</ol>
		</li>
		<li>Remove the prehybridization solution, and transfer the coverslips to a humidified chamber.
		<ol>
			<li>Pipette 30-50 &#181;L of probe solution onto parafilm to form individual droplets.</li>
		</ol>
		</li>
		<li>Incubate the samples overnight (12-18 h) at 37 &#176;C. 
		 
		DAY 2</li>
		<li>Transfer the coverslips back into a 12-well plate, and remove excess probe solution by washing for 4 x 5 min with 400 &#181;L of probe wash buffer at 37 &#176;C. 
		&#8203;NOTE: As an alternative to placing 30-50 &#181;l aliquots onto parafilm and under coverslips for incubation, add 300 &#181;L of probe solution directly to coverslips in a 12-well plate. This procedure is simpler, but requires substantial amounts of reagents. Heat the probe wash buffer to 37 &#176;C before use. Calculate the amount of buffer needed, and transfer it to a 15 mL conical centrifuge tube.</li>
		<li>Wash samples for 2 x 5 min with 5x SSCT at RT.</li>
		<li>Replace the 5x SSCT solution with 1x PBS, and store the samples at 4 &#176;C until amplification.</li>
	</ol>
	</li>
	<li>Amplification
	<ol>
		<li>Remove the 1x PBS solution from the wells, and add at least 300 &#181;L of amplification buffer to each well. Incubate the samples for 30 min at RT.</li>
		<li>Prepare each HCR hairpin (h1 and h2) by snap-cooling the desired volume in separate tubes.
		<ol>
			<li>To prepare 300 &#181;L of amplification solution, use 18 pmol of each hairpin (e.g., for 300 &#181;L, use 6 &#181;L of a 3 &#181;M stock hairpin solution).</li>
			<li>Transfer the hairpin solution into tubes.</li>
			<li>Incubate at 95 &#176;C for 90 s.</li>
			<li>Cool to RT for 30 min in the dark.</li>
		</ol>
		</li>
		<li>Prepare a hairpin mixture by adding the snap-cooled &#34;h1&#34; and &#34;h2&#34; hairpins to the amplification buffer.</li>
		<li>Place drops of 30-50 &#181;L of hairpin mixture onto parafilm.</li>
		<li>Incubate the samples overnight (12-18 h) in the dark at RT. 
		 
		DAY 3</li>
		<li>Transfer the coverslips back into the 12 well plate, and remove excess hairpins by washing for 5 x 5 min with 5x SSCT at RT with agitation.</li>
		<li>Aspirate the 5x SSCT buffer, and replace it with 1x PBS. 
		NOTE: If required, use the RNA-FISH samples in a standard immunofluorescence assay, followed by staining of nuclei (see section 5).</li>
	</ol>
	</li>
	<li>Nuclear staining and slide mounting
	<ol>
		<li>Aspirate the 1x PBS solution, and replace it with 4&#8242;,6-diamidino-2-phenylindole (DAPI, 0.2 &#181;g/mL) in 1x PBS solution.</li>
		<li>Incubate the samples for 10 min at RT in the dark.</li>
		<li>Aspirate the DAPI solution, and wash the cells twice with 1x PBS.</li>
		<li>Place two drops of 10 &#181;L each of mounting medium; ensure that the drops are separated sufficiently to allow two coverslips to be placed on a single slide.</li>
		<li>Remove excess liquid by tapping the coverslips on a clean towel, and then place them in antifade mounting medium with the cells facing down.</li>
		<li>Place the mounted samples on a dry, flat surface in the dark and let them cure.</li>
		<li>Following curing, seal the edges of the coverslips with VALAP sealant or nail polish to prevent the samples from drying out.</li>
	</ol>
	</li>
</ol>
5. SARS-CoV-2 RNA-FISH in HAE cultures
DAY 1
<ol start="1">
	<li>Fixing and permeabilizing the HAE culture
	<ol>
		<li>Aspirate the medium, and fix the infected cells using 3.7% PFA solution for 1 h at RT.</li>
		<li>Aspirate the 3.7% PFA solution, and wash the cells twice with 1x PBS.
		<ol>
			<li>Replace the 3.7% PFA solution with 1x PBS under a Transwell insert.</li>
		</ol>
		</li>
		<li>Discard the PBS, and dehydrate the samples using 2 x 5 min washes with 100% MeOH prechilled to -20 &#176;C.</li>
		<li>After the second wash, replace the buffer with fresh, chilled MeOH for permeabilization under the Transwell insert. Store overnight at -20 &#176;C.</li>
	</ol>
	</li>
</ol>
DAY 2
<ol start="2">
	<li>Rehydration 
	&#8203;Rehydrate the samples through a graded series of MeOH/PBST solutions (each for 5 min) on ice: 75% MeOH/25% PBST, 50% MeOH/50% PBST, 25% MeOH/75% PBTS, and 100% PBST (twice).
	<ol>
		<li>Wash the cells for 5 min on ice with 50% 5x SSCT/50% PBST.</li>
		<li>Wash cells for 5 min on ice with 5x SSCT.</li>
		<li>Replace the 5x SSCT buffer with 1x PBS.</li>
	</ol>
	</li>
	<li>Detection
	<ol>
		<li>Incubate the cells (inside the Transwell insert) for 5 min on ice with 100 &#181;L of probe hybridization buffer. Next, transfer the plate to incubator for 30 min at 37 &#176;C (prehybridization). 
		NOTE: The probe hybridization buffer must be pre-heated to 37 &#176;C before use. Calculate the required volume: 100 &#181;L is needed for a single Transwell insert.</li>
		<li>Prepare the probe solution. As 1 mL of probe solution requires 4 pmol of probe, add 4 &#181;L of 1 &#181;M probe stock solution to 1 mL of probe hybridization buffer, and mix well. 
		&#8203;NOTE: For RNA detection, use 100 &#181;L of probe solution per Transwell insert. Leave the probe solution on ice until the end of the prehybridization step.</li>
		<li>Remove the prehybridization solution, and add the probe solution.</li>
		<li>Incubate the cells overnight (12-18 h) at 37 &#176;C. 
		 
		&#8203;DAY 3</li>
		<li>Remove excess probe by washing for 4 x 15 min with 100 &#181;L of probe wash buffer at 37 &#176;C.</li>
		<li>Wash the samples for 2 x 5 min with 5x SSCT at RT.</li>
		<li>Replace the 5x SSCT with 1x PBS, and store the samples at 4 &#176;C until amplification.</li>
	</ol>
	</li>
	<li>Amplification
	<ol>
		<li>Preamplify the samples by incubating them with amplification buffer for 30 min at RT.</li>
		<li>Prepare each hairpin by snap-cooling the desired volumes in separate tubes.
		<ol>
			<li>To prepare 500 &#181;L of amplification solution, use 30 pmol of each hairpin (e.g., for 500 &#181;L, use 10 &#181;L of 3 &#181;M stock hairpin solution).</li>
			<li>Transfer the hairpin solution to the tubes.</li>
			<li>Incubate at 95 &#176;C for 90 s.</li>
			<li>Cool to RT for 30 min in the dark.</li>
		</ol>
		</li>
		<li>Prepare the hairpin solution by adding all snap-cooled hairpins to 500 &#181;L of amplification buffer at RT.</li>
		<li>Remove the preamplification solution, and add the complete hairpin solution.</li>
		<li>Incubate the samples overnight (12-18 h) at RT in the dark.</li>
		<li>Remove excess hairpins by washing with 5x SSCT at RT as follows: 2 x 5 min, 2 x 15 min, and 1 x 5 min.</li>
		<li>Replace the 5x SSCT solution with 1x PBS, and store at 4 &#176;C for not more than 2-3 days or proceed directly to nuclear staining. 
		&#8203;NOTE: If required, use the RNA-FISH samples in a standard immunofluorescence assay, followed by nuclear staining (see section 6).</li>
	</ol>
	</li>
	<li>Nuclear staining and slide mounting
	<ol>
		<li>Aspirate the 1x PBS solution, and replace it with DAPI solution (0.2 &#181;g/mL) in 1x PBS.</li>
		<li>Incubate the samples for 10 min at RT in the dark.</li>
		<li>Aspirate the DAPI solution, and wash the cells twice with 1x PBS.</li>
		<li>Place the cut-out membrane from the Transwell inserts onto 10 &#181;L of antifade mounting medium with the cells facing up, and add extra mounting medium (5 &#181;L) to the membrane.</li>
		<li>Cover the membranes with coverslips.</li>
		<li>Cure the mounted samples on a dry, flat surface in the dark.</li>
		<li>Following curing, seal the edges of the coverslips with VALAP sealant or nail polish to prevent the samples from drying out.</li>
	</ol>
	</li>
</ol>
6. Immunofluorescence analysis of Vero cells and HAE cultures
NOTE: Perform the immunofluorescence assay on day 3 for cell lines or day 4 for HAE cultures. Use a different approach for each model. All differences are indicated clearly.
<ol>
	<li>Aspirate the 1x PBS solution from the wells.</li>
	<li>Block the samples by incubation with 1% w/v bovine serum albumin in PBST solution for 30 min at 37 &#176;C.</li>
	<li>Prepare primary antibodies by preparing appropriate dilutions in blocking solution, and incubate.
	<ol>
		<li>For Vero cells on coverslips:
		<ol>
			<li>Place drops (30-50 &#181;L) of antibody solution onto parafilm in a humidity chamber.</li>
			<li>Place coverslips onto the antibody drops with the cells facing down.</li>
		</ol>
		</li>
		<li>For HAE, replace the blocking agent in the inserts with antibody solution (100 &#181;L), and incubate the samples in a humidified chamber. 
		NOTE: Adjust the time and temperature for each incubation with primary antibody. Typical parameters are 2&#160;h at RT or overnight at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Wash the samples for 3 x 5 min with PBST.
	<ol>
		<li>For cells on coverslips, transfer to a 12-well plate and add PBST solution.</li>
		<li>For HAE cultures, replace the antibody solution with PBST solution.</li>
	</ol>
	</li>
	<li>Check the light sources and filters available for the confocal microscope.</li>
	<li>Choose secondary antibodies according to the host species. Choose the fluorophore parameters (excitation and emission wavelengths, spectrum width, and excitation efficiency according to the available light source). 
	NOTE: Spectral parameters can be modeled using online tools (see Table of Materials).</li>
	<li>Prepare appropriate secondary antibody solutions by diluting them with blocking solution.</li>
	<li>Incubate with secondary antibodies (as in 6.3), but incubate for 1 h at 37 &#176;C.</li>
	<li>Wash the samples as in step 6.4.</li>
	<li>Nuclear staining and slide mounting
	<ol>
		<li>For cells on coverslips
		<ol>
			<li>Aspirate the PBST, and replace it with DAPI (0.2 &#181;g/mL) in 1x PBS solution.</li>
			<li>Incubate the samples for 10 min at RT in the dark.</li>
			<li>Aspirate the DAPI solution, and wash the cells twice with 1x PBS.</li>
			<li>Place the coverslips onto drops of 10 &#181;L of mounting medium with the cells facing down.</li>
			<li>Seal the coverslips with nail polish.</li>
		</ol>
		</li>
		<li>For HAE cultures
		<ol>
			<li>Aspirate the 1x PBST, and replace it with DAPI (0.2 &#181;g/mL) in 1x PBS solution.</li>
			<li>Incubate the samples for 10 min at RT in the dark.</li>
			<li>Aspirate the DAPI solution, and wash the cells twice with 1x PBS.</li>
			<li>Place the cut-out membranes onto drops of 10 &#181;L of mounting medium with the cells facing up, and add extra (5 &#181;L) mounting medium to the membrane.</li>
			<li>Cover the membranes with coverslips.</li>
			<li>Seal the coverslips with nail polish.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
7. Confocal microscopy
<ol>
	<li>Define the tracks by specifying the fluorophores used.</li>
	<li>Choose the scanning mode and speed.</li>
	<li>Adjust the laser power, gain, and offset values for each fluorophore by comparing them with respective negative controls: for virus, mock-infected cells; for cellular proteins, samples stained with isotype control antibodies from an appropriate host.</li>
	<li>To acquire a 3D image, activate z-stack mode, and set the top and bottom limits. Set the step size/number. 
	NOTE: For more details on coronavirus imaging, see19.</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Well-differentiated+human+airway+epithelial+cell+cultures.">Well-differentiated human airway epithelial cell cultures.</a> Human Cell Culture Protocols in Methods in Molecular Medicine. 107, 183-206 (2005).</li><li>Milewska, A., Owczarek, K., Szczepanski, A., Pyrc, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+coronavirus+entry+into+cells.">Visualizing coronavirus entry into cells.</a> Methods in Molecular Biology. 2203, 241-261 (2020).</li></ol>

The authors have no conflicts of interest to declare.

Immuno-RNA-FISH is a reliable method for double-staining of RNA and cellular proteins. Here, a modified immuno-RNA-FISH protocol has been described that allows detection of SARS-CoV-2 RNA and cellular proteins in cell lines and HAE cultures. This protocol can be adapted for use in different cell models including cell monolayers or specific tissues. The method relies on the concept of an HCR initiated by appropriate probe localization. Next, the use of split-initiator probes to begin amplification of the signal by fluores...

SARS-CoV-2 is a novel human betacoronavirus that emerged at the end of 2019, causing an unprecedented pandemic a few months later. Because the virus is new to science, much of its biology and its impact on host cells remain unknown. Therefore, mapping the virus-cell and -tissue tropism during infection is important if its basic biological characteristics and its effects on the host are to be understood. Several techniques are used to examine virus-host interplay including biochemical, biological, and physical assays. In situ hybridization is a common method that employs labeled complementary DNA, RNA, or modified nucleic acid probes, which localize to specific DNA or RNA sequences in a cell or tissue.
A new RNA fluorescent in situ hybridization (RNA-FISH) method has been developed that incorporates modifications to increase sensitivity by amplifying the signal-to-noise ratio via an HCR1. HCR allows the study of RNA localization at a single-cell level. Owing to its high specificity, sensitivity, and resolution, this method is useful not only for basic science studies, but also for applicatory projects, e.g., diagnostics. Recently, the feasibility of this method was demonstrated for detecting SARS-CoV-2 RNA localized to ciliated cells within fully differentiated 3D human airway epithelium (HAE) cultures2. HAE cultures constitute one of the most advanced tools used to study viral infection in the context of the &#34;natural infection&#34; microenvironment3,4.
Several reports on human coronaviruses (HCoV), including SARS-CoV-2, highlight the importance of epigenetic modifications with respect to HCoV infection and pathophysiology [reviewed in 5], e.g., the methylation pattern of the gene encoding the angiotensin-converting enzyme 2 (ACE-2) receptor6,7. Interestingly, mass-spectrometric screening identified several epigenetic factors that interact with the SARS-CoV-2 proteome8. More specifically, nonstructural protein 5 (NSP5) binds to the epigenetic regulator, histone deacetylase 2, and the catalytically inactive NSP5 (C145A) interacts with tRNA methyltransferase 1 (24). Additionally, NSP16 methyltransferase activity is blocked by the methyltransferase inhibitor, sinefungin9. However, the exact role of these epigenetic factors in COVID-19 remains unclear. Replication of HCoV takes place in the cytoplasm of the infected cell, and triggers inflammatory responses that are regulated by epigenetic modifications10.
For instance, HCoV-229E fine-tunes nuclear factor-kappa B signaling and profoundly reprograms the host cellular chromatin landscape by increasing acetylation of H3K36 and H4K5 in certain regions11. The Middle East respiratory syndrome-related coronavirus infection increases levels of H3K27me3 and depletes H3K4me3 at the promoter regions of subsets of specific interferon-sensitive genes12. Additionally, viral RNA triggers cell immune responses, as demonstrated for flaviviruses13, retroviruses14,15, and coronaviruses16. The epigenetic markers on viral RNA may play a role in recognition by cellular sensors, as shown for m7A methylation of human immunodeficiency virus-1 RNA17. However, questions remain: What is the impact of SARS-CoV-2 RNA on the immune response, and are epigenetic marks involved?
Here, an optimized RNA-FISH method coupled with immunofluorescence analysis of cell lines and 3D tissues (fully differentiated HAE) has been described. Although cytological methods, such as FISH and immunofluorescence, are used widely, this new-generation in situ hybridization method based on HCR has never been used for virus detection (except in a recent publication)2. In general, immunostaining and FISH require the following steps: permeabilization to enable penetration of probes or antibodies; fixation in which cellular material is fixed and preserved; detection in which antibodies or nucleic acid probes are applied; and finally, mounting of the samples for visualization.
Although existing protocols share these general features, they vary markedly with respect to the parameters involved. Here, an optimized, simple, immuno-RNA-FISH protocol has been described to detect SARS-CoV-2 RNA in HAE cultures and Vero cells. The technique comprises the following steps: (1) fixation of cells with paraformaldehyde; (2) permeabilization with detergent or methanol (MeOH); (3) rehydration through a graded series of MeOH solutions (HAE cultures only); (4) detection; (5) amplification using HCR technology to detect SARS-CoV-2 RNA; (6) immunostaining; and (7) imaging under a confocal microscope.

<table><tbody><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>Confocal Microscope LSM 880</td><td>ZEISS </td><td></td><td></td></tr><tr><td>Grant Bio, Mini Rocker- Shaker</td><td>Fisher Scientific</td><td>12965501</td><td></td></tr><tr><td>Incubator Galaxy170R</td><td>New Brunswick</td><td>CO170R-230-1000</td><td></td></tr><tr><td>Thermomixer Comfort</td><td>Eppendorf</td><td>5355 000 011</td><td></td></tr><tr><td>Materials</td><td></td><td></td><td></td></tr><tr><td>15 mm x 15 mm NO. 1 coverslips</td><td>LabSolute</td><td>7695022</td><td></td></tr><tr><td>1.5 mL tubes</td><td>FL-MEDICAL</td><td>5.350.023.053</td><td></td></tr><tr><td>12-well plate</td><td>TTP</td><td>92412</td><td></td></tr><tr><td>Conical centrifuge tube</td><td>Sarstedt</td><td>5.332.547.254</td><td></td></tr><tr><td>parafilm</td><td>Sigma</td><td>P7793-1EA</td><td></td></tr><tr><td>serological pipets</td><td>VWR Collection</td><td>612-5523P, 612-5827P</td><td></td></tr><tr><td>slide glass</td><td>PTH CHEMLAND</td><td>04-296.202.03</td><td></td></tr><tr><td>Transwell ThinCerts</td><td>Grainer bio-one</td><td>665641</td><td></td></tr><tr><td>Reagents</td><td></td><td></td><td></td></tr><tr><td>Alexa fluorophore 488-conjugated secondary antibodies </td><td>Invitrogen</td><td></td><td></td></tr><tr><td>&amp;beta;5-tubulin </td><td>Santa Cruz Biotechnology</td><td> sc-134234</td><td></td></tr><tr><td>DAPI</td><td>Thermo Scientific</td><td>D1306</td><td></td></tr><tr><td>Disodium phosphate</td><td>Sigma</td><td>S51136-500G</td><td></td></tr><tr><td>EGTA</td><td>BioShop</td><td>EGT101.25</td><td></td></tr><tr><td>HCR Amplification Buffer</td><td>Molecular Instruments, Inc.</td><td>BAM01522</td><td>Buffer can be also prepared doi:10.1242/dev.165753: Supplementary information</td></tr><tr><td>HCR amplifier B1-h1 Alexa Fluor 647</td><td>Molecular Instruments, Inc.</td><td>S013922</td><td></td></tr><tr><td>HCR amplifier B1-h2 Alexa Fluor 647</td><td>Molecular Instruments, Inc.</td><td>S012522</td><td></td></tr><tr><td>HCR Probe Hybridization Buffer</td><td>Molecular Instruments, Inc.</td><td>BPH03821</td><td>Buffer can be also prepared doi:10.1242/dev.165753: Supplementary information</td></tr><tr><td>HCR probe set for SARS-CoV-2 Ncapsid</td><td>Molecular Instruments, Inc.</td><td>PRE134</td><td></td></tr><tr><td>HCR Probe Wash Buffer</td><td>Molecular Instruments, Inc.</td><td>BPW01522</td><td>Buffer can be also prepared doi:10.1242/dev.165753: Supplementary information</td></tr><tr><td>HEPES</td><td>BioShop</td><td>HEP001.100</td><td></td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Sigma</td><td>63138-250G</td><td></td></tr><tr><td>Methanol</td><td>Sigma</td><td>32213-1L-M</td><td></td></tr><tr><td>Monopotassium phosphate</td><td>Sigma</td><td>P5655-100G</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148-1KG</td><td></td></tr><tr><td>PIPES</td><td>BioShop</td><td>PIP666.100</td><td></td></tr><tr><td>Potassium Chloride</td><td>Sigma</td><td>P5405-250G</td><td></td></tr><tr><td>Prolong Diamond Antifade Mounting Medium</td><td>Invitrogen</td><td>P36970</td><td></td></tr><tr><td>Sodium Chloride</td><td>BioShop</td><td>SOD001.5</td><td></td></tr><tr><td>Trisodium Citrate 2-hydrate</td><td>POCH</td><td> 6132-04-3</td><td></td></tr><tr><td>Tween-20</td><td>BioShop</td><td>TWN580.500</td><td></td></tr><tr><td></td><td>Software</td><td></td><td></td></tr><tr><td>Fluorescence Spectraviewer</td><td></td><td></td><td>Modeling spectral parameters</td></tr><tr><td>ImageJ Fiji</td><td></td><td></td><td>Acquiring and processing z-stack images</td></tr></tbody></table>

visualization of sars-cov-2 using immuno rna-fluorescence in situ hybridization

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in cell line&#160;and three-dimensional (3D) cultures of human airway epithelium. The method allows highly specific and sensitive visualization of viral RNA by relying on HCR initiated by probe localization. Split-initiator probes help amplify the signal by fluorescently labeled amplifiers, resulting in negligible background fluorescence in confocal microscopy. Labeling amplifiers with different fluorescent dyes facilitates the simultaneous recognition of various targets. This, in turn, allows the mapping of the infection in tissues to better understand viral pathogenesis and replication at the single-cell level. Coupling this method with immunofluorescence may facilitate better understanding of host-virus interactions, including alternation of the host epigenome and immune response pathways. Owing to sensitive and specific HCR technology, this protocol can also be used as a diagnostic tool. It is also important to remember that the technique may be modified easily to enable detection of any RNA, including non-coding RNAs and RNA viruses that may emerge in the future.

The immuno-RNA-FISH protocol described in this manuscript was carried out using two cellular systems: a Vero cell line and a 3D HAE culture. The major steps for both cellular models are shown in Table 2. The RNA-FISH protocol for visualization of SARS-CoV-2 in HAE cultures includes steps that are typical for tissue samples, i.e., permeabilization with 100% MeOH and rehydration through a graded series of MeOH-PBS and 0.1% Tween solutions. Immunofluorescence was performed a...

Watch this Scientific Journal Video about Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization at JoVE.com

Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

Here, we describe a simple method that combines RNA fluorescence in situ&#160;hybridization (RNA-FISH) with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. This protocol may increase understanding of the molecular characteristics of SARS-CoV-2 RNA-host interactions at a single-cell level.

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory ...

visualization-sars-cov-2-using-immuno-rna-fluorescence-situ

Research

JoVE Journal

Immunology and Infection

6.0K Views.  Jagiellonian University. Hier beschrijven we een eenvoudige methode die RNA-fluorescentie in situ hybridisatie (RNA-FISH) combineert met immunofluorescentie om ernstig acuut respiratoir syndroom coronavirus 2 (SARS-CoV-2) RNA te visualiseren. Dit protocol kan het begrip van de moleculaire kenmerken van SARS-CoV-2 RNA-host interacties op eencellig niveau vergroten.

Video: Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

Next-generation Viral RNA/DNA in situ Hybridization Applications in Human Immunodeficiency Virus/Simian Immunodeficiency Virus Research

In situ hybridization is a powerful technique to identify specific RNA or DNA sequences within individual cells in tissue sections, providing important insights into physiological processes and disease pathogenesis. In situ hybridization (ISH) has been used for many years to assess the location of cells infected by viruses, but recently a next-generation ISH approach was developed with a unique probe design strategy that allows simultaneous signal amplification and background suppression to achieve single-molecule visualization while preserving tissue morphology. This next-generation ISH is based on an approach like branched PCR, but performed in situ and is more facile, sensitive, and reproducible than classical ISH methods or in situ PCR approaches in routinely detecting RNA or DNA in formalin-fixed paraffin embedded (FFPE) tissues. For the last several years our laboratory has been applying this ISH platform for the detection of human immunodeficiency (HIV) and simian immunodeficiency (SIV) viral RNA (vRNA) and/or viral DNA (vDNA) positive cells within a multitude of FFPE tissues. With this detailed technical manuscript, we would like to share our knowledge and advice with all individuals interested in using next-generation ISH in their research.

Here we present a next-generation in situ hybridization assay to identify specific viral RNA or DNA sequences in formalin-fixed paraffin embedded (FFPE) tissues. This approach allows visualization of low copies of RNA and DNA in less than 24 h with very high sensitivity and specificity.

In situ hybridization is a powerful technique to identify specific RNA or DNA sequences within individual cells in tissue sections, providing important ...

Genetics

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

FISH for Long Non-Coding RNA Localization in Human Osteosarcoma Cells

RNA Fluorescence In Situ Hybridization for Long Non-Coding RNA Localization in Human Osteosarcoma Cells

The important roles of long non-coding RNAs (lncRNAs) in cancer have been studied, such as regulating the proliferation, epithelial-mesenchymal transition (EMT), migration, infiltration, and autophagy of cancer cells. Localization detection of lncRNAs in cells can provide insight into their functions. By designing the lncRNA-specific antisense chain sequence followed by labeling with fluorescent dyes, RNA fluorescence in situ hybridization (FISH) can be applied to detect the cellular localization of lncRNAs. Together with the development of microscopy, the RNA FISH techniques now even allow for visualization of the poorly expressed lncRNAs. This method can not only detect the localization of lncRNAs alone, but also detect the colocalization of other RNAs, DNA, or proteins by using double-color or multicolor immunofluorescence. Here, we have included the detailed experimental operation procedure and precautions of RNA FISH by using lncRNA small nucleolar RNA host gene 6 (SNHG6) in human osteosarcoma cells (143B) as an example, to provide a reference for researchers who want to perform RNA FISH experiments, especially lncRNA FISH.

Author Spotlight: RNA FISH for Locating lncRNA-SNHG6 in Osteosarcoma Cells 

The present protocol describes a method of RNA fluorescence in situ hybridization to localize the lncRNAs in human osteosarcoma cells.

The important roles of long non-coding RNAs (lncRNAs) in cancer have been studied, such as regulating the proliferation, epithelial-mesenchymal transition ...

Cancer Research

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...

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 ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the presence of neutralizing antibodies against the virus may provide protection against future infection. Thus, as the creation and translation of effective COVID-19 vaccines continues at an unprecedented speed, the development of fast and effective methods to measure neutralizing antibodies against SARS-CoV-2 will become increasingly important to determine long-term protection against infection for both previously infected and immunized individuals. This paper describes a high-throughput protocol using vesicular stomatitis virus (VSV) pseudotyped with the SARS-CoV-2 spike protein to measure the presence of neutralizing antibodies in convalescent serum from patients who have recently recovered from COVID-19. The use of a replicating pseudotyped virus eliminates the necessity for a containment level 3 facility required for SARS-CoV-2 handling, making this protocol accessible to virtually any containment level 2 lab. The use of a 96-well format allows for many samples to be run at the same time with a short turnaround time of 24 h.

The protocol described here outlines a fast and effective method for measuring neutralizing antibodies against the SARS-CoV-2 spike protein by evaluating the ability of convalescent serum samples to inhibit infection by an enhanced green fluorescent protein-labeled vesicular stomatitis virus pseudotyped with spike glycoprotein.

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the ...

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 ...

High-throughput Confocal Imaging of Quantum Dot-Conjugated SARS-CoV-2 Spike Trimers to Track Binding and Endocytosis in HEK293T Cells

The development of new technologies for cellular fluorescence microscopy has facilitated high-throughput screening methods for drug discovery. Quantum dots are fluorescent nanoparticles with excellent photophysical properties imbued with bright and stable photoluminescence as well as narrow emission bands. Quantum dots are spherical in shape, and with the proper modification of the surface chemistry, can be used to conjugate biomolecules for cellular applications. These optical properties, combined with the ability to functionalize them with biomolecules, make them an excellent tool for investigating receptor-ligand interactions and cellular trafficking. Here, we present a method that uses quantum dots to track the binding and endocytosis of SARS-CoV-2 spike protein. This protocol can be used as a guide for experimentalists looking to utilize quantum dots to study protein-protein interactions and trafficking in the context of cellular physiology.

In this protocol, quantum dots conjugated to recombinant SARS-CoV-2 spike enable cell-based assays to monitor spike binding to hACE2 at the plasma membrane and subsequent endocytosis of the bound proteins into the cytoplasm.

The development of new technologies for cellular fluorescence microscopy has facilitated high-throughput screening methods for drug discovery. Quantum ...

Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

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