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. …

Divulgaciones

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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Using Immuno-RNA Fluorescence In Situ Hybridization to Visualize SARS-CoV-2. J. Vis. Exp. (Pending Publication), e21907, doi: (2024).

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