Plaque Assay–Based Quantification of Viral Load in an Infected Mouse Tissue Sample

Take epithelial cell monolayers. Add diluted supernatants from a virus-infected mouse tissue homogenate.

Incubate for viral adsorption, then wash.

Cover the cells with agar-containing medium, forming a semisolid overlay, and incubate.

The virus enters cells, replicates, and lyses them, releasing new viruses.

The solidified agar restricts viral spread, forming clear zones called plaques.

Remove the overlay. Treat with a fixative to inactivate viruses.

Add a permeabilization buffer to disrupt cell membranes.

Add a blocking buffer to prevent non-specific interactions.

Add primary antibodies. Incubate to allow their interactions with viral nucleocapsid proteins.

Wash and add biotin-conjugated secondary antibodies that bind to the primary antibodies.

Rinse and introduce avidin-biotin-peroxidase complexes, which bind to biotin on the secondary antibodies.

Add chromogenic substrates that react with peroxidase to produce brown precipitates, which stain the plaques.

Count the plaques at different dilutions to quantify the viral load in the tissue sample.

Utilizing the supernatant obtained from the tissue homogenates, perform tenfold serial dilution and infect confluent monolayers of Vero E6 cells with o