JoVE 実験百科事典
微生物学
0 回視聴 • 4:42 分 • July 31st, 2026
Take a coverslip containing a permeabilized, virus-infected epithelial cell monolayer.
The virus forms double-stranded RNA (dsRNA) intermediates, which are coated with nucleoproteins. These intermediates are detected by cytoplasmic pattern recognition receptors (PRRs).
Add a cocktail of primary antibodies diluted in a blocking reagent. Incubate.
The dsRNA-specific antibody detects viral dsRNA, while the PRR-specific antibody binds the host cytoplasmic receptor.
Wash with a buffer.
Add green- and red-fluorophore-tagged secondary antibodies and incubate to bind the corresponding primary antibodies. Wash.
Add magenta fluorophore-tagged antibodies and incubate to directly detect the viral nucleoproteins. Wash.
Add DAPI to stain the nuclei.
Introduce a nonionic detergent to remove nonspecifically bound fluorophore-tagged antibodies.
Wash with buffer and water to remove detergent residues.
Mount the coverslip in an antifade mounting medium and seal.
Under confocal microscopy, observe the colocalization of red fluorescence with green and magenta signals, confirming the interaction between viral dsRNA and host cytoplasmic PRRs.
After completed incubation, aspirate the medium and rinse the cells with 1
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