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

Summary

Here, we describe a simple method that combines RNA fluorescence in situ 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.

Abstract

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 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.

Introduction

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 "natural infection" microenvironment^3,4.

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) receptor^6,7. 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, retroviruses^14,15, 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.

Protocol

1. Buffer preparation

1. For 500 mL of 2x PHEM buffer, combine 18.14 g of piperazine-N,N′-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 °C until required.
   NOTE: The buffer will not be clear until the pH reaches 7.0.
2. 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 µL 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 °C until required.
   NOTE: Formaldehyde should be handled in a fume hood while wearing protective gloves and a lab coat.
3. 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 °C until required.
   NOTE: After thawing, the buffer can be stored at 4 °C for up to 3 months.
4. Prepare PBST (0.1% Tween-20 in 1x phosphate-buffered saline [PBS]). For 50 mL, add 50 µL of 100% Tween-20 to 50 mL of 1x PBS and mix well.
   NOTE: The solution can be stored at room temperature (RT).
5. 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.
6. 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 µm filter into an autoclaved bottle.
7. For 50 mL of 5x SSCT, combine 12.5 mL of 20x SSC buffer with 37.5 mL of dH₂O, and add 50 µL of 100% Tween-20. Mix well.
8. For 50 mL of 50% 5x SSCT/50% PBST, combine 25 mL of 5x SSCT with 25 mL of PBST.
9. For 50 mL of 2x SSC, combine 5 mL of 10x SSC buffer with 45 mL of dH₂O. Mix well.
   ​NOTE: All SSC buffers should be stored at RT in the dark.

2. Target definition, probes, and amplifiers

1. Use the tools available on the manufacturer'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).
2. Determine the best probe set size (a set of 20 probes is sufficient for visualization).
3. Set the amplifiers used for target RNA detection.
   1. 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, …) for each target RNA to be imaged within the same sample (e.g., amplifier B1 for target 1, amplifier B2 for target 2, …).

3. Cell culture and infection with SARS-CoV-2

1. Culture Vero (monkey kidney epithelial cells) cells in Dulbecco's modified Eagle medium containing 5% fetal bovine serum.
   1. Seed 50,000 cells onto coverslips (No. 1, 15 × 15 mm) in a 12-well plate.
2. 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.
3. Virus infection
   1. Inoculate cells with SARS-CoV-2 at 1000x median tissue culture infectious dose (TCID₅₀) per mL
   2. Incubate cells for 2 h at 37 °C.
   3. Wash cells twice with PBS to remove unbound virus.
   4. Culture cells for 48 h.

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

DAY 1

1. Fixing and permeabilizing cells
   1. Fix infected cells with 3.7% w/v PFA solution for 1 h at RT.
   2. Aspirate the 3.7% PFA solution, and wash the cells twice using 1x PBS.
   3. Permeabilize the cells with PBST solution for 10 min at RT with agitation.
   4. Aspirate the PBST, and wash the cells twice with 1x PBS.
2. Detection
   1. Aspirate the 1x PBS solution, and wash the cells twice with 2x SSC at RT.
   2. Aspirate the 2x SSC solution, and prehybridize the samples by adding at least 300 µL of probe hybridization buffer. Cover the wells containing the cells, and incubate at 37 °C for 30 min.
   3. Prepare the probe solution by adding 1.2 pmol of probe mixture to the probe hybridization buffer.
      1. Use 1.2 µL of the 1 µM probe stock to prepare 300 µL of working stock.
   4. Remove the prehybridization solution, and transfer the coverslips to a humidified chamber.
      1. Pipette 30-50 µL of probe solution onto parafilm to form individual droplets.
   5. Incubate the samples overnight (12-18 h) at 37 °C.
      
      DAY 2
   6. Transfer the coverslips back into a 12-well plate, and remove excess probe solution by washing for 4 x 5 min with 400 µL of probe wash buffer at 37 °C.
      ​NOTE: As an alternative to placing 30-50 µl aliquots onto parafilm and under coverslips for incubation, add 300 µL 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 °C before use. Calculate the amount of buffer needed, and transfer it to a 15 mL conical centrifuge tube.
   7. Wash samples for 2 x 5 min with 5x SSCT at RT.
   8. Replace the 5x SSCT solution with 1x PBS, and store the samples at 4 °C until amplification.
3. Amplification
   1. Remove the 1x PBS solution from the wells, and add at least 300 µL of amplification buffer to each well. Incubate the samples for 30 min at RT.
   2. Prepare each HCR hairpin (h1 and h2) by snap-cooling the desired volume in separate tubes.
      1. To prepare 300 µL of amplification solution, use 18 pmol of each hairpin (e.g., for 300 µL, use 6 µL of a 3 µM stock hairpin solution).
      2. Transfer the hairpin solution into tubes.
      3. Incubate at 95 °C for 90 s.
      4. Cool to RT for 30 min in the dark.
   3. Prepare a hairpin mixture by adding the snap-cooled "h1" and "h2" hairpins to the amplification buffer.
   4. Place drops of 30-50 µL of hairpin mixture onto parafilm.
   5. Incubate the samples overnight (12-18 h) in the dark at RT.
      
      DAY 3
   6. 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.
   7. 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).
4. Nuclear staining and slide mounting
   1. Aspirate the 1x PBS solution, and replace it with 4′,6-diamidino-2-phenylindole (DAPI, 0.2 µg/mL) in 1x PBS solution.
   2. Incubate the samples for 10 min at RT in the dark.
   3. Aspirate the DAPI solution, and wash the cells twice with 1x PBS.
   4. Place two drops of 10 µL each of mounting medium; ensure that the drops are separated sufficiently to allow two coverslips to be placed on a single slide.
   5. Remove excess liquid by tapping the coverslips on a clean towel, and then place them in antifade mounting medium with the cells facing down.
   6. Place the mounted samples on a dry, flat surface in the dark and let them cure.
   7. Following curing, seal the edges of the coverslips with VALAP sealant or nail polish to prevent the samples from drying out.

5. SARS-CoV-2 RNA-FISH in HAE cultures

DAY 1

1. Fixing and permeabilizing the HAE culture
   1. Aspirate the medium, and fix the infected cells using 3.7% PFA solution for 1 h at RT.
   2. Aspirate the 3.7% PFA solution, and wash the cells twice with 1x PBS.
      1. Replace the 3.7% PFA solution with 1x PBS under a Transwell insert.
   3. Discard the PBS, and dehydrate the samples using 2 x 5 min washes with 100% MeOH prechilled to -20 °C.
   4. After the second wash, replace the buffer with fresh, chilled MeOH for permeabilization under the Transwell insert. Store overnight at -20 °C.

DAY 2

2. Rehydration
   ​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).
   1. Wash the cells for 5 min on ice with 50% 5x SSCT/50% PBST.
   2. Wash cells for 5 min on ice with 5x SSCT.
   3. Replace the 5x SSCT buffer with 1x PBS.
3. Detection
   1. Incubate the cells (inside the Transwell insert) for 5 min on ice with 100 µL of probe hybridization buffer. Next, transfer the plate to incubator for 30 min at 37 °C (prehybridization).
      NOTE: The probe hybridization buffer must be pre-heated to 37 °C before use. Calculate the required volume: 100 µL is needed for a single Transwell insert.
   2. Prepare the probe solution. As 1 mL of probe solution requires 4 pmol of probe, add 4 µL of 1 µM probe stock solution to 1 mL of probe hybridization buffer, and mix well.
      ​NOTE: For RNA detection, use 100 µL of probe solution per Transwell insert. Leave the probe solution on ice until the end of the prehybridization step.
   3. Remove the prehybridization solution, and add the probe solution.
   4. Incubate the cells overnight (12-18 h) at 37 °C.
      
      ​DAY 3
   5. Remove excess probe by washing for 4 x 15 min with 100 µL of probe wash buffer at 37 °C.
   6. Wash the samples for 2 x 5 min with 5x SSCT at RT.
   7. Replace the 5x SSCT with 1x PBS, and store the samples at 4 °C until amplification.
4. Amplification
   1. Preamplify the samples by incubating them with amplification buffer for 30 min at RT.
   2. Prepare each hairpin by snap-cooling the desired volumes in separate tubes.
      1. To prepare 500 µL of amplification solution, use 30 pmol of each hairpin (e.g., for 500 µL, use 10 µL of 3 µM stock hairpin solution).
      2. Transfer the hairpin solution to the tubes.
      3. Incubate at 95 °C for 90 s.
      4. Cool to RT for 30 min in the dark.
   3. Prepare the hairpin solution by adding all snap-cooled hairpins to 500 µL of amplification buffer at RT.
   4. Remove the preamplification solution, and add the complete hairpin solution.
   5. Incubate the samples overnight (12-18 h) at RT in the dark.
   6. 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.
   7. Replace the 5x SSCT solution with 1x PBS, and store at 4 °C for not more than 2-3 days or proceed directly to nuclear staining.
      ​NOTE: If required, use the RNA-FISH samples in a standard immunofluorescence assay, followed by nuclear staining (see section 6).
5. Nuclear staining and slide mounting
   1. Aspirate the 1x PBS solution, and replace it with DAPI solution (0.2 µg/mL) in 1x PBS.
   2. Incubate the samples for 10 min at RT in the dark.
   3. Aspirate the DAPI solution, and wash the cells twice with 1x PBS.
   4. Place the cut-out membrane from the Transwell inserts onto 10 µL of antifade mounting medium with the cells facing up, and add extra mounting medium (5 µL) to the membrane.
   5. Cover the membranes with coverslips.
   6. Cure the mounted samples on a dry, flat surface in the dark.
   7. Following curing, seal the edges of the coverslips with VALAP sealant or nail polish to prevent the samples from drying out.

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.

1. Aspirate the 1x PBS solution from the wells.
2. Block the samples by incubation with 1% w/v bovine serum albumin in PBST solution for 30 min at 37 °C.
3. Prepare primary antibodies by preparing appropriate dilutions in blocking solution, and incubate.
   1. For Vero cells on coverslips:
      1. Place drops (30-50 µL) of antibody solution onto parafilm in a humidity chamber.
      2. Place coverslips onto the antibody drops with the cells facing down.
   2. For HAE, replace the blocking agent in the inserts with antibody solution (100 µL), and incubate the samples in a humidified chamber.
      NOTE: Adjust the time and temperature for each incubation with primary antibody. Typical parameters are 2 h at RT or overnight at 4 °C.
4. Wash the samples for 3 x 5 min with PBST.
   1. For cells on coverslips, transfer to a 12-well plate and add PBST solution.
   2. For HAE cultures, replace the antibody solution with PBST solution.
5. Check the light sources and filters available for the confocal microscope.
6. 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).
7. Prepare appropriate secondary antibody solutions by diluting them with blocking solution.
8. Incubate with secondary antibodies (as in 6.3), but incubate for 1 h at 37 °C.
9. Wash the samples as in step 6.4.
10. Nuclear staining and slide mounting
    1. For cells on coverslips
       1. Aspirate the PBST, and replace it with DAPI (0.2 µg/mL) in 1x PBS solution.
       2. Incubate the samples for 10 min at RT in the dark.
       3. Aspirate the DAPI solution, and wash the cells twice with 1x PBS.
       4. Place the coverslips onto drops of 10 µL of mounting medium with the cells facing down.
       5. Seal the coverslips with nail polish.
    2. For HAE cultures
       1. Aspirate the 1x PBST, and replace it with DAPI (0.2 µg/mL) in 1x PBS solution.
       2. Incubate the samples for 10 min at RT in the dark.
       3. Aspirate the DAPI solution, and wash the cells twice with 1x PBS.
       4. Place the cut-out membranes onto drops of 10 µL of mounting medium with the cells facing up, and add extra (5 µL) mounting medium to the membrane.
       5. Cover the membranes with coverslips.
       6. Seal the coverslips with nail polish.

7. Confocal microscopy

1. Define the tracks by specifying the fluorophores used.
2. Choose the scanning mode and speed.
3. 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.
4. 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.

Results

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...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
Equipment
Confocal Microscope LSM 880	ZEISS
Grant Bio, Mini Rocker- Shaker	Fisher Scientific	12965501
Incubator Galaxy170R	New Brunswick	CO170R-230-1000
Thermomixer Comfort	Eppendorf	5355 000 011
Materials
15 mm x 15 mm NO. 1 coverslips	LabSolute	7695022
1.5 mL tubes	FL-MEDICAL	5.350.023.053
12-well plate	TTP	92412
Conical centrifuge tube	Sarstedt	5.332.547.254
parafilm	Sigma	P7793-1EA
serological pipets	VWR Collection	612-5523P, 612-5827P
slide glass	PTH CHEMLAND	04-296.202.03
Transwell ThinCerts	Grainer bio-one	665641
Reagents
Alexa fluorophore 488-conjugated secondary antibodies	Invitrogen
β5-tubulin	Santa Cruz Biotechnology	sc-134234
DAPI	Thermo Scientific	D1306
Disodium phosphate	Sigma	S51136-500G
EGTA	BioShop	EGT101.25
HCR Amplification Buffer	Molecular Instruments, Inc.	BAM01522	Buffer can be also prepared doi:10.1242/dev.165753: Supplementary information
HCR amplifier B1-h1 Alexa Fluor 647	Molecular Instruments, Inc.	S013922
HCR amplifier B1-h2 Alexa Fluor 647	Molecular Instruments, Inc.	S012522
HCR Probe Hybridization Buffer	Molecular Instruments, Inc.	BPH03821	Buffer can be also prepared doi:10.1242/dev.165753: Supplementary information
HCR probe set for SARS-CoV-2 Ncapsid	Molecular Instruments, Inc.	PRE134
HCR Probe Wash Buffer	Molecular Instruments, Inc.	BPW01522	Buffer can be also prepared doi:10.1242/dev.165753: Supplementary information
HEPES	BioShop	HEP001.100
Magnesium sulfate heptahydrate	Sigma	63138-250G
Methanol	Sigma	32213-1L-M
Monopotassium phosphate	Sigma	P5655-100G
Paraformaldehyde	Sigma	P6148-1KG
PIPES	BioShop	PIP666.100
Potassium Chloride	Sigma	P5405-250G
Prolong Diamond Antifade Mounting Medium	Invitrogen	P36970
Sodium Chloride	BioShop	SOD001.5
Trisodium Citrate 2-hydrate	POCH	6132-04-3
Tween-20	BioShop	TWN580.500
	Software
Fluorescence Spectraviewer			Modeling spectral parameters
ImageJ Fiji			Acquiring and processing z-stack images