Immunofluorescent Detection of Viral Infection in Murine Epidermal Whole Mounts

Begin with a mouse epidermal skin sheet infected with herpes simplex virus, with the basal side facing the medium.

The infected basal keratinocytes contain early viral proteins.

Remove the medium. Add a fixative to preserve tissue morphology and protein localization.

Wash with phosphate buffer to remove residual fixative.

Add a detergent-supplemented blocking buffer to prevent nonspecific interactions while the detergent permeabilizes the cells.

Introduce primary antibodies against viral proteins and keratin, a marker of basal keratinocytes, then incubate.

Wash with a detergent-containing buffer to remove unbound antibodies.

Add fluorescently labeled secondary antibodies and a nuclear dye. The secondary antibodies bind to the primary antibodies, and the nuclear dye stains the nuclei blue.

Wash with a detergent-containing buffer.

Mount the epidermal sheet with a coverslip, so that its basal keratinocytes face the objective lens for microscopic observation.

The green viral proteins are visible within the red keratinocytes, showing viral infection in the keratinocytes.

To perform HSV-1 wild type infection of an epidermal sheet in one well of a 24-well plate, calculate the virus titer based on th