Visualization of Viral Double-Stranded RNA and Host Pattern Recognition Receptors in Infected Epithelial Cells

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 mL PBS supplemented with calcium and magnesium per well.

After removing the PBS, add 1 mL of -20°C pre-chilled methanol per well and incubate at -20°C for 15 minutes to fix the cells.

Then remove methanol and add 1 mL of PBS to each well.

Wash samples at 4°C for 5 minutes with gentle rocking, and repeat this wash for a total of four times.

Wash the cells further with 1 mL of 0.2% t-octylphenoxypolyethoxyethanol per well at 4°C for 5 minutes with gentle rocking.

Then remove it and add 1 mL of PBS to each well and wash samples at 4°C for 5 minutes with gentle rocking, and repeat again for a total of four times.

To detect double-stranded RNA and MDA5, add 200 µL of primary antibodies diluted in 3% BSA and incubate with gentle rocking at 4°C overnight.

Then remove primary antibodies and wash each well with 1 mL of PBS at room temperature for 5 minutes with gentle rocking, and repeat the wash four more times.

Then add 200 µL of secondary antibodies diluted in 3% BSA, and incubate covered at room temperature for 1 hour with gentle rocking.

After removing the secondary antibodies, wash each well with 1 mL of PBS for 5 minutes with gentle rocking at room temperature, and repeat the wash four more times.

Then counterstain the coverslips by adding DAPI and incubating at room temperature for 3 minutes with gentle rocking.

Wash the coverslips with 1 mL of 0.5% t-octylphenoxypolyethoxyethanol per well, three times, 5 minutes, with gentle rocking at room temperature.

Wash with 1 mL of PBS per well for 5 minutes twice, with gentle rocking at room temperature.

Then wash once in 1 mL of double-distilled H₂O per well, rocking gently for 1 minute at room temperature.

After completed staining and washing, mount the coverslips onto glass slides using one drop of mounting medium and incubate overnight at room temperature, covered in the dark.

Then seal the slides with nail polish and air dry for 1 hour.

Image the samples using confocal microscope with the 60x magnification and 1.42 numerical aperture oil immersion lens, making sure to use the same laser emissions for each sample, and then proceed with analysis.