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

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.