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

Abstract

Source: Wei Liu et al., Merkel Cell Polyomavirus Infection and Detection, J. Vis. Exp. (2019).

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 co....

Materials

List of materials used in this article

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