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.

For immunostaining of the brain tissue sections, wash the samples two times for 1 hour in 4 milliliters of 0.2% Triton X-100 in PBS, followed by permeabilization for two days at 37 degrees Celsius, in 4 milliliters of 0.2% Triton X-100, 20% dimethylsulfoxide, and 0.3 molar glycine in PBS. Block any nonspecific binding with 4 milliliters of 0.2% Triton X-100, 10% dimethylsulfoxide, and 6% normal serum in PBS for two days at 37 degrees Celsius.

At the end of the blocking incubation, label the samples with 2 milliliters of primary antibody solution for five days at 37 degrees Celsius, refreshing the antibody solution after two and half days. Next, wash the samples for one day in 4 milliliters of 0.2% Tween 20 and 10 micrograms per milliliter heparin in PBS, exchanging the wash buffer at least four to five times during the course of the day and leaving the final wash on overnight.

The next day, incubate the samples in 2 milliliters of secondary antibody solution for five days at 37 degrees Celsius. Then, wash the samples in 4 milliliters of fresh 0.2% Tween 20 and 10 micrograms per milliliter heparin in PBS as demonstrated.

For nuclear staining of the samples, incubate the samples in 4 milliliters of the nucleic acid stain TO-PRO-3 for 5 hours, protected from light. At the end of the incubation, wash the samples in 0.2% Tween 20 and 10 micrograms per milliliter heparin in PBS as demonstrated, followed by dehydration in an ascending tert-butanol series for 2 hours per immersion.

After the 90% immersion, transfer the samples to a 96% tert-butanol solution overnight. The next morning, dehydrate the samples further in 100% tert-butanol for 2 hours before clearing the tissue sections in freshly prepared BABB-D15 for 2 to 6 hours until they are optically transparent. The samples can then be stored in BABB-D15, protected from light until their mounting and imaging.

For sample mounting, use a 3D printer to print an imaging chamber and lid. Mount a 30-millimeters diameter coverslip onto the imaging chamber with one component room temperature vulcanizing silicone rubber. Similarly, mount a 22-millimeters coverslip onto the lid. Use a water wetted cotton swab to remove the excess silicone rubber on both rings before allowing the rubber to cure overnight.

The next day, place a sample in the imaging chamber and add a small volume of BABB-D15 to the chamber before inserting the lid. Using a hypodermic needle, fill the chamber up with BABB-D15 through the inlet and plug the inlet before sealing the imaging chamber with silicone rubber.

To set up the image acquisition, select the appropriate laser lines for the fluorophores used and adjust the detection ranges of each detector to prevent signal overlap between channels. Then set the acquisition parameters, define the upper and lower border of the stack, and acquire the image stack.

To generate 3D projections of the image stack, open the image files in Fiji and select Image, Color, Channels tool, More, and Split channels to split the merged images into individual channels. For bleach correction of the images for each channel, select Image, Adjust, Bleach Correction, and select Simple ratio. Use the sliders to adjust the brightness and contrast for each channel and select Image, Color, and Merge Channels. Tick the option Create composite.

Convert the image to RGB format and select Image, Stacks, and 3D project to generate a 3D projection. Select brightest point as the projection method and set the slice spacing to match the Z-step size of the acquired image stack. For maximum quality, set the rotation angle increment to 1 and enable interpolation. Modify the Total rotation, Transparency thresholds, and Opacity as needed. Then save the 3D projection as both .TIFF and .AVI file formats.