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

Protocol

1. Infection

1. Maintain primary human dermal fibroblasts in DMEM with 10% fetal calf serum, 1% non-essential amino acids, and 1% L-glutamine. Upon reaching confluence, split fibroblasts 1:4 without spinning down.
   NOTE: For the highest MCPyV infection efficiency, use primary fibroblasts between passages 5 and 12 that are actively dividing at the time of plating.
2. To infect human dermal fibroblasts, aspirate the medium and wash the cells with DPBS.
3. Add 1 mL of 0.05% Trypsin-EDTA to the dish and incubate at 37 °C for 5-10 min.
4. Check under the microscope to make sure that the cells are coming off the dish.
5. Add 10 mL of DMEM/F12 medium containing 20 ng/mL EGF, 20 ng/mL bFGF, and 3 µM CHIR99021, all of which stimulate MCPyV infection, to the dish and transfer the cell solution into a 15 mL tube.
6. Spin down the cells at 180 x g for 2 min and discard the supernatant. Resuspend the cells in DMEM/F12 medium containing 20 ng/mL EGF, 20 ng/mL bFGF and 3µM CHIR99021 at 2-4 x 10⁴ cells per mL.
7. Seed 200 µL of the cell suspension supplemented with 1 mg/mL collagenase type IV into each well of a 96-well plate. Thaw MCPyV virion stock on ice. Add 10⁹ viral genome equivalents of MCPyV virions per 1 µL of iodixanol for each 2,500 to 5,000 cells to be infected.  Tap the side of the plate gently and place the plate in the incubator.
8. After incubation at 37 °C in 5% CO₂ for 48-72 h, add 20% FBS to each well.
9. Allow the infection to proceed at 37 °C in 5% CO₂ for 72-96 h.

2. In situ DNA-HCR

1. Fix cells cultured on coverslips with 4% PFA in PBS for 10 min. Wash the coverslips twice with PBS, then treat them with 70% ethanol to permeabilize the cells at 4 °C overnight.
   NOTE: Fixed samples can remain in this state for several days.
2. Pre-hybridize samples by replacing ethanol with probe hybridization buffer and incubating for 60 min at RT.
3. Dilute the probe(s) (1 µM) (Table 1) in probe hybridization buffer solution at 1:500 dilution 30 min prior to the end of the pre-hybridization step and incubate at 45 °C.
4. At the end of the incubations, pipette ~10 μL of the diluted probe mix per coverslip on microscope slides. Place the coverslips, cell-side down, on their respective droplets of the hybridization probe mix. Seal the edges and backs of the coverslips to the slides with a liberal amount of rubber cement.
5. Heat the slides with added probes at 94 °C for 3 min by setting the slides on the flat side of a heat block. Transfer the slides to a humidified chamber and incubate at 45 °C overnight. To make a simple humidified chamber, lightly dampen sterile paper towels with dH₂O and place them in the bottom of a plastic container that seals with a rubber gasket.
   NOTE: During heating, the rubber cement can bubble briefly. However, ensure that the seal does not break.
6. Carefully peel away the rubber cement with forceps and place the coverslips cell-side up back into wells of a 24-well plate. Wash the coverslips with probe wash buffer at RT three times.
7. Incubate the coverslip in the amplification buffer (200 µL in 24-well plates) at RT for 30-60 min.
8. Meanwhile, anneal each of the two labeled oligonucleotide hairpins recognizing the probes (Table 1) in separate PCR tubes by heating to 94 °C for 90 s and cooling to RT for 30 min while protecting from light. Mix the two hairpins in amplification buffer, each at a dilution of 1:50.
9. Make a surface for the amplification reaction by stretching paraffin film over the open face of a 24-well plate lid. Pipette 50-100 µL droplets of the hairpin/amplification buffer mixture onto the paraffin film for each coverslip. Use forceps to carefully remove the coverslips from the pre-amplification solution. Dry the coverslip by touching the edge to a porous disposable wipe, and place cell-side down onto the amplification droplet.
10. Place the plate lid holding the coverslips into a humidified chamber and incubate overnight at RT in the dark.
11. Return the coverslips to wells once more. Incubate the samples with 5x SSCT [5x sodium chloride sodium citrate (SSC) 0.1% Tween 20 filtered through a 0.22 µm filter] containing 0.5 µg/mL DAPI for 1 h at RT. Wash the samples twice with 5x SSCT at RT.
12. Mount the coverslips on microscope slides. Analyze the cells and image the samples using an inverted fluorescence microscope.

MCPyV probe 1	CCTCAACCTACCTCCAACTCTCACCATATTCGCTTC TAAAA tgagctacctcactaaggagtggtttttatactgcagtttcccgcccttg ATTTT CACATTTACAGACCTCAACCTACCTCCAACTCTCAC
MCPyV probe 2	CCTCAACCTACCTCCAACTCTCACCATATTCGCTTC TAAAA agaggcctcggaggctaggagccccaagcctctgccaacttgaaaaaaaa ATTTT CACATTTACAGACCTCAACCTACCTCCAACTCTCAC
MCPyV probe 3	CCTCAACCTACCTCCAACTCTCACCATATTCGCTTC TAAAA cattgactcatttcctggagaggcggagtttgactgataaacaaaacttt ATTTT CACATTTACAGACCTCAACCTACCTCCAACTCTCAC
MCPyV probe 4	CCTCAACCTACCTCCAACTCTCACCATATTCGCTTC TAAAA gatactgccttttttgctaattaagcctcttaagcctcagagtcctctct ATTTT CACATTTACAGACCTCAACCTACCTCCAACTCTCAC
MCPyV probe 5	CCTCAACCTACCTCCAACTCTCACCATATTCGCTTC TAAAA aagcttctcctgtaagaatagcttccaaagttactcctgtggtggcactt ATTTT CACATTTACAGACCTCAACCTACCTCCAACTCTCAC
MCPyV probe 6	CCTCAACCTACCTCCAACTCTCACCATATTCGCTTC TAAAA ggatgttgccataacaattaggagcaatctccaaaagcttgcacagagcc ATTTT CACATTTACAGACCTCAACCTACCTCCAACTCTCAC
MCPyV probe 7	CCTCAACCTACCTCCAACTCTCACCATATTCGCTTC TAAAA gctcaggggaggaaagtgattcatcgcagaagagatcctcccaggtgcca ATTTT CACATTTACAGACCTCAACCTACCTCCAACTCTCAC
MCPyV probe 8	CCTCAACCTACCTCCAACTCTCACCATATTCGCTTC TAAAA aagcctgggacgctgagaaggacccatacccagaggaagagctctggctg ATTTT CACATTTACAGACCTCAACCTACCTCCAACTCTCAC
MCPyV probe 9	CCTCAACCTACCTCCAACTCTCACCATATTCGCTTC TAAAA agcttcgggaccccccaaattttcgctttcttgagaatggaggaggggtc ATTTT CACATTTACAGACCTCAACCTACCTCCAACTCTCAC
MCPyV probe 10	CCTCAACCTACCTCCAACTCTCACCATATTCGCTTC TAAAA cttttggctagaacagtgtctgcggcttgttggcaaatggttttctgaga ATTTT CACATTTACAGACCTCAACCTACCTCCAACTCTCAC
HPV probe 1	CCTCAACCTACCTCCAACTCTCACCATATTCGCTTC TAAAA cagctctgtgcataactgtggtaactttctgggtcgctcctgtgggtcct ATTTT CACATTTACAGACCTCAACCTACCTCCAACTCTCAC
HPV probe 2	CCTCAACCTACCTCCAACTCTCACCATATTCGCTTC TAAAA acaatattgtaatgggctctgtccggttctgcttgtccagctggaccatc ATTTT CACATTTACAGACCTCAACCTACCTCCAACTCTCAC
HPV probe 3	CCTCAACCTACCTCCAACTCTCACCATATTCGCTTC TAAAA gtcagctatactgggtgtaagtccaaatgcagcaatacaccaatcgcaac ATTTT CACATTTACAGACCTCAACCTACCTCCAACTCTCAC
HPV probe 4	CCTCAACCTACCTCCAACTCTCACCATATTCGCTTC TAAAA ctttggtatgggtcgcggcggggtggttggccaagtgctgcctaataatt ATTTT CACATTTACAGACCTCAACCTACCTCCAACTCTCAC
HPV probe 5	CCTCAACCTACCTCCAACTCTCACCATATTCGCTTC TAAAA ccatccattacatcccgtaccctcttccccattggtacctgcaggatcag ATTTT CACATTTACAGACCTCAACCTACCTCCAACTCTCAC

Table 1: Probes used in the study.

Materials

Name	Company	Catalog Number	Comments
Probe hybridization buffer	Molecular technologies
Probe wash buffer	Molecular technologies
Amplification buffer	Molecular technologies
Alexa 594-labeled hairpins	Molecular technologies	B4	Protect from light
Paraformaldehyde	Sigma	P6148