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

Take fixed and permeabilized coronavirus-infected Vero cells — an interferon-deficient mammalian cell line.

Inside the host, viral RNA is present as subgenomic or sgRNAs, intermediate-length RNA molecules encoding specific viral proteins synthesized via transcription.

Add hybridization chain reaction or HCR initiator probes.

The HCR probe binds to its complementary sequence on the target RNA.

Introduce fluorescently labeled oligonucleotide hairpin probes, H-1 and H-2.

The initiator probe binds to its complementary H1 tail, linearizing the hairpin structure.

The exposed H1 sequence binds to its complementary H2 tail.

The trailing H2 sequence continues binding to an H1 tail, amplifying fluorescent-tagged probes around the target area, and generating a robust signal.

Add appropriate antibodies to visualize the host cell cytoskeleton.

Stain the nuclei using DAPI.

Under a fluorescence microscope, virus-infected cells display red fluorescence in the cytoplasm, indicating the presence of the target RNA.

After fixing and permeabilizing the cells, as described in the text manuscript, remove the 1x PBS solution from the wells, and add at least 300 microliters of amplification buffer to each well. Incubate the samples for 30 minutes at room temperature. Prepare each HCR hairpin by snap-cooling the desired volume in separate tubes.

To prepare 300 microliters of amplification solution, use 18 picamoles of each hairpin. Transfer the hairpin solution into tubes, and incubate them at 95 degrees Celsius for 90 seconds. Then, cool to room temperature for 30 minutes in the dark. Prepare a hairpin mixture by adding the snap-cooled H1 and H2 hairpins to the amplification buffer. Place drops of 30 to 50 microliters of hairpin mixture onto parafilm, and incubate the samples overnight in the dark at room temperature.

To mount the slides, place two 10-microliter drops of mounting medium on a slide, ensuring that the drops are separated sufficiently to allow two coverslips to be placed on a single slide. Remove excess liquid by tapping the coverslips on a clean towel. Then, place them in anti-fade mounting medium with the cells facing down. Place the mounted samples on a dry flat surface in the dark and let them cure.