HCR-DNA FISH: A Fluorescence In Situ Hybridization Technique to Detect Viral DNA in Infected Cells
Published: April 30, 2023
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 videodan 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 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. To infect human dermal fibroblasts, aspirate the medium and wash the cells with DPBS. Add 1…
Açıklamalar
The authors have nothing to disclose.
Materials
| 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 |
Bu Makaleden Alıntı Yapın
HCR-DNA FISH: A Fluorescence In Situ Hybridization Technique to Detect Viral DNA in Infected Cells. J. Vis. Exp. (Pending Publication), e20353, doi: (2023).