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