Three-Dimensional Visualization of a Rabies Virus Infection in Mouse Brain Tissue

Take a fixed thick brain section from a mouse infected with rabies virus.

The virus infects neurons and replicates, resulting in viral proteins present within the cells.

Treat with a detergent to permeabilize cellular membranes and a blocking solution to mask non-specific binding sites.

Incubate with primary antibodies targeting the viral proteins, followed by fluorophore-conjugated secondary antibodies that bind to the primary antibodies.

Treat with a fluorescent nucleic acid-binding dye to label the nucleus.

Dehydrate the tissue in increasing alcohol concentrations, then apply a clearing solution to increase tissue transparency for imaging.

Mount the section in an imaging chamber, fill it with the clearing solution, and seal it.

Using confocal laser scanning microscopy, image the tissue separately using the fluorescent labels, then merge them to create a two-dimensional image.

Acquire images at different focal planes across the tissue and combine them to generate a three-dimensional representation for evaluating infection progression.