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.

Use a fluorescence microscope to detect the bright fluorescence from the replication foci within the nuclei of infected cells. In the following protocol, we will show in situ HCR-DNA FISH to detect Merkel Cell Polyomavirus genome in infected primary human skin cells.

- First, fix the cells onto cover slips with 4% PFA in PBS for 10 minutes. Then, wash the cover slips twice with PBS and treat them with 70% ethanol at 4 degrees Celsius overnight to permeablize the cells. The next day, replace the ethanol with probe hydration buffer and incubate at room temperature for 60 minutes to pre-hybridize the samples. 30 minutes before the end of the pre-hybridization incubation, dilute the probes in probe hybridization solution at a 1:500 dilution and incubate at 45 degrees Celsius.

When the incubations are complete, pipette approximately 10 microliters of the diluted probe mixture onto a microscope slide for each cover slip. Place each cover slip, cell-side down, on its respective droplet of hybridization mix. Using a liberal amount of rubber cement, seal the edges and back of each cover slip to its slide. Set the slides on the flat side of a heat block and heat them to 94 degrees Celsius for 3 minutes. After this, transfer the slides to a humidified chamber and incubate them at 45 degrees Celsius overnight.

The next day, use forceps to carefully peel away the rubber cement. Place the cover slips, cell-side up, into the wells of a 24-well plate and wash them with probe wash buffer three times at room temperature. Add 200 microliters of amplification buffer to each well containing a cover slip and incubate at room temperature for 30 to 60 minutes. Meanwhile, anneal each of the two labeled oligonucleotide hairpins recognizing the probes in separate PCR tubes by heating them to 95 degrees Celsius for 90 seconds and then cooling them to room temperature for 30 minutes. Mix the hairpins together in amplification buffer at a dilution of 1:50.

Next, stretch paraffin film over the open face of a 12-well plate lid to make a surface for the amplification reaction. Pipette 50 to 100 microliters droplets of the hairpin mixture onto the paraffin film for each cover slip. Using forceps, carefully remove each cover slip from the pre-amplification solution and touch the edge to a porous, disposable wipe to dry. Place each dried cover slip, cell-side down, onto an amplification droplet. Transfer the plate lid into a humidified chamber and incubate overnight at room temperature and in the dark.

The next day, return the cover slips to the wells of the 24-well plate. Add 5x SSCT-containing DAPI at a concentration of 0.5 micrograms per milliliter to each well and incubate at room temperature for one hour. After this, wash the samples twice with 5x SSCT at room temperature. Mount the washed cover slips onto microscope slides, and use an inverted fluorescence microscope to analyze the cells and image the samples.