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

Overview

In this video, the cells infected with MCPyV virions were subjected to in situ hybridization chain reaction to detect MCPyV specific genomes. Further, the DNA-HCR technique was combined with FISH to visualize the MCPyV DNA in infected human skin 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