This protocol enables the visualization of virus infection and provides deep insights into spatiotemporal resolution of the infection environment and its surrounding cellular context. The applicability of immunostaining to deep tissue imaging not only allows the detection of viruses, but in fact also enables the differentiation of various cellular subpopulations using their respective cell markers. To fix the tissue for immunostaining place the brain samples in a one to 10 ratio of 4%paraformaldehyde in PBS to sample tissue volume for at least 48 hours at four degrees Celsius.
At the end of the fixation period wash the tissue samples three times in PBS for at least 30 minutes per wash before transferring the samples to 02%sodium azide in PBS at four degrees Celsius. Then use a vibratome set to a 0.3 to 0.5 millimeters per second blade feed rate to section the tissues into one-millimeter thick sections. Store the sections in fresh 02%sodium azide in PBS at four degrees Celsius.
For methanol pretreatment immerse the samples in an ascending methanol series as indicated for one hour per incubation at room temperature with gentle oscillation. After the last incubation cool the samples to four degrees Celsius. Replace the 100%methanol with four milliliters of pre-chilled bleaching solution for an overnight incubation at four degrees Celsius.
The next morning replace the bleaching solution with four milliliters of fresh 80%methanol for a one hour incubation at room temperature. Then immerse the samples in a descending series of methanol in reverse of what was just demonstrated, washing the samples one time for one hour in four milliliters of PBS after the 20%methanol immersion. For immunostaining of the brain tissue sections wash the samples two times for one hour in four milliliters of 0.2%Triton X-100 in PBS, followed by permeabilization for two days at 37 degrees Celsius in four milliliters of 0.2%Triton X-100, 20%dimethyl sulfoxide, and 0.3 molar glycine in PBS.
Block any nonspecific binding with four milliliters of 0.2%Triton X-100, 10%dimethyl sulfoxide, and 6%normal serum in PBS for two days at 37 degrees Celsius. At the end of the blocking incubation label the samples with two milliliters of primary antibody solution for five days at 37 degrees Celsius, refreshing the antibody solution after two and 1/2 days. Next, wash the samples for one day in four 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 two milliliters of secondary antibody solution for five days at 37 degrees Celsius. Then wash the samples in four 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 four milliliters of the nucleic acid stain TO-PRO-3 for five 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 two 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 two hours before clearing the tissue sections in freshly prepared BABB-D15 for two to six 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-millimeter diameter coverslip onto the imaging chamber with one-component room temperature vulcanizing silicone rubber.
Similarly, mount a 22-millimeter 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 to find the upper and lower border of the Z-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 a maximum quality set the Rotation angle increment to one 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.
Using immunostaining of rabies virus phosphoprotein complex layers of infected neuronal cells can be visualized in thick sections of mouse brain tissue samples. Subsequently, seamless 3D projections of the acquired image stacks can be reconstructed. Because of the high resolution with which the image stacks are acquired, the infection can be assessed up to a single cell level, allowing assertions about, for example, the abundance and distribution of antigen within a cell of interest.
In addition to mouse brain tissue, the protocol can be applied to brain tissue samples from other animal species. For example, sections obtained from different compartments of an infected ferret brain reveal a varying degree of rabies virus infection. Astrocytes can be differentiated via the expression of glial fibrillary acid protein while neurites can be specifically stained for microtubule-associated protein II.Simultaneously, viral proteins can be co-stained to assess the relationship between the infected cells and the highlighted cellular subpopulation.
For immunostaining the choice of antibodies to detect the proteins of interest is crucial. While standard immunohistochemistry concentrations are often a good starting point, some antibodies may need additional adjustment. Many of the chemicals used in this protocol have harmful properties.
Conduct the respective experiment in a fume hood wearing appropriate personal protective equipment including a lab coat and gloves.
