HCR-DNA FISH: A Fluorescence In Situ Hybridization Technique to Detect Viral DNA in Infected Cells

- HCR-FISH, or hybridization chain reaction-fluorescence in situ hybridization, detects viral nucleic acid sequences within the genome of virus-infected cells. To begin, fix an adherent culture of virus-infected cells and permeablize their cell membrane. Incubate the cells with a hybridization buffer to reduce any background signal. Treat the cells with initiator probes carrying an HCR initiator sequence and a complementary sequence targeting the viral DNA. Heat the sample to denature the host double-stranded DNA to single-stranded DNA. Incubate the sample to allow the probe to bind to its complimentary target viral DNA sequence.

Next, treat with an amplification buffer containing two sets of fluorescent-labeled oligonucleotide hairpin probes, H1 and H2. Incubate to facilitate the hybridization chain reaction. The unpaired initiator sequence binds to its complementary H1 tail, linearizing the hairpin structure. The exposed H1 sequence similarly binds to its complementary H2 tail. The trailing H2 continues to bind to an H1 tail. This way, the fluorescent-tagged probes amplify around the target site, emitting a strong signal. Treat the sample with DAPI to stain the cell nuclei.

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