Detection of Zika Viral dsRNA Replication Intermediates Using Immunocytochemistry

Begin with a multiwell plate containing epithelial cells infected with the Zika virus, a single-stranded RNA virus.

During infection, the virus releases the RNA that forms a double-stranded RNA or dsRNA intermediate.

To detect these dsRNA intermediates, replace the medium with methanol to permeabilize the cells and preserve cellular integrity.

Remove the methanol and wash with a buffer.

Add a blocking solution to prevent non-specific antibody binding.

Add a primary antibody specific to the viral dsRNA.

Incubate with gentle rocking at a low temperature to allow the primary antibody to bind to the viral dsRNA.

Rinse with a buffer, add a red fluorophore-conjugated secondary antibody and incubate.

The secondary antibodies bind to the primary antibodies.

Wash with a buffer, then add a fluorescent DNA-binding dye to stain the nucleus.

Observe under a fluorescence microscope to visualize the labeled viral dsRNA intermediate within the infected cell.

For immunocytochemistry, remove the medium from the wells of the 12-well plate, and add one milliliter of methanol to each well. Allow the cells to fix at four degrees Celsius for 30 minutes. Next, wash the fixed cells three times with 1x PBS. Then add b