Using Immuno-RNA Fluorescence In Situ Hybridization to Visualize SARS-CoV-2
Published: January 31, 2024
Abstract
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.
Protocol
1. SARS-CoV-2 RNA-FISH in Vero cells cultured on coverslips DAY 1 Fixing and permeabilizing cells Fix infected cells with 3.7% w/v PFA solution for 1 h at RT. Aspirate the 3.7% PFA solution, and wash the cells twice using 1x PBS. Permeabilize the cells with PBST solution for 10 min at RT with agitation. Aspirate the PBST, and wash the cells twice with 1x PBS. …
Declarações
The authors have nothing to disclose.
Materials
| 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 |
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Citar este artigo
Using Immuno-RNA Fluorescence In Situ Hybridization to Visualize SARS-CoV-2. J. Vis. Exp. (Pending Publication), e21907, doi: (2024).