Source: Kula-Pacurar, A., et al. Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization. J. Vis. Exp. (166), (2020).
This video demonstrates the application of Hybridization Chain Reaction (HCR)-Fluorescence In Situ Hybridization (FISH) for visualizing the spatial distribution of SARS-CoV-2 subgenomic RNA. Combining HCR with immunofluorescence techniques has the potential to provide insights into host-virus interactions at the single-cell level.
1. SARS-CoV-2 RNA-FISH in Vero cells cultured on coverslips
DAY 1
DAY 2
3. Amplification
DAY 3
4. Nuclear staining and slide mounting
2. SARS-CoV-2 RNA-FISH in HAE cultures
DAY 1
DAY 2
DAY 3
5. Nuclear staining and slide mounting
3. Immunofluorescence analysis of Vero cells and HAE cultures
NOTE: Perform the immunofluorescence assay on day 3 for cell lines or day 4 for HAE cultures. Use a different approach for each model. All differences are indicated clearly.
4. Confocal microscopy
The authors have nothing to disclose.
Equipment | |||
Confocal Microscope LSM 880 | ZEISS | ||
Incubator Galaxy170R | New Brunswick | CO170R-230-1000 | |
Materials | |||
15 mm x 15 mm NO. 1 coverslips | LabSolute | 7695022 | |
1.5 mL tubes | FL-MEDICAL | 5.350.023.053 | |
12-well plate | TTP | 92412 | |
Alexa fluorophore 488-conjugated secondary antibodies | Invitrogen | ||
β5-tubulin | Santa Cruz Biotechnology | sc-134234 | |
DAPI | Thermo Scientific | D1306 | |
HCR Amplification Buffer | Molecular Instruments, Inc | BAM01522 | Buffer can be also prepared doi:10.1242/dev.165753: Supplementary information |
HCR amplifier B1-h1 Alexa Fluor 647 | Molecular Instruments, Inc. | S013922 | |
HCR amplifier B1-h2 Alexa Fluor 647 | Molecular Instruments, Inc. | S012522 | |
HCR Probe Hybridization Buffer | Molecular Instruments, Inc. | BPH03821 | Buffer can be also prepared doi:10.1242/dev.165753: Supplementary information |
HCR probe set for SARS-CoV-2 Ncapsid | Molecular Instruments, Inc. | PRE134 | |
HCR Probe Wash Buffer | Molecular Instruments, Inc. | BPW01522 | Buffer can be also prepared doi:10.1242/dev.165753: Supplementary information |
Fluorescence Spectraviewer | Modeling spectral parameters | ||
ImageJ Fiji | Acquiring and processing z-stack images | ||
ImmunoFluorescence (IF) | Blocking | (1% BSA in PBST) 30 min at 37 °C | |
Primary antibody incubation | (Antibody solution of apropriate concentration in blocking solution) 2 h at room temperature / overnight at 4 °C, 30-50 µL | (Antibody solution of apropriate concentration in blocking solution) 2 h at room temperature / overnight at 4 °C, 100 µL | |
Primary antibody washing | (PBST) 3 x 5 min at room temperature | ||
Secondary antibody incubation | (Antibody solution of apropriate concentration in blocking solution) 1 h at 37 °C, 30-50 µL | (Antibody solution of appropriate concentration in blocking solution) 1 h at 37 °C, 100 µL | |
Secondary antibody washing | (PBST) 3 x 5 min at room temperature | ||
Nuclear staining | (DAPI solution) 10 min at room temperature, then 2 x 5 min with 1x PBS |