The authors thank Thomas C. Mettenleiter and Verena te Kamp for critically reading the manuscript. This work was supported by the Federal Excellence Initiative of Mecklenburg Western Pomerania and the European Social Fund (ESF) Grant KoInfekt (ESF/14-BM-A55-0002/16) and an intramural collaborative research grant on Lyssaviruses at the Friedrich-Loeffler-Institute (Ri-0372).

RABV-infected, PFA-fixed archived brain material was used. The respective animal experimental studies were evaluated by the responsible animal care, use, and ethics committee of the State Office for Agriculture, Food Safety, and Fishery in Mecklenburg-Western Pomerania (LALFF M-V) and gained approval with permissions 7221.3-2.1-002/11 (mice) and 7221.3-1-068/16 (ferrets). General care and methods used in the animal experiments were carried out according to the approved guidelines.
CAUTION: This protocol uses various toxic and/or harmful substances, including PFA, methanol (MeOH), hydrogen peroxide (H2O2), sodium azide (NaN3), TBA, BA, BB, and DPE. MeOH and TBA are highly flammable. Avoid exposure by wearing appropriate personal protective equipment (a lab coat, gloves, and eye protection) and conducting experiments in a fume hood. Collect waste separately in appropriate containers and dispose of it according to local regulations. Rabies virus is classified as a biosafety level (BSL)-2 pathogen and can, therefore, generally be handled under BSL-2 conditions. Some activities, including procedures that may generate aerosols, work with high virus concentrations, or work with novel lyssaviruses, may require BSL-3 classification. Pre-exposure prophylaxis is recommended for high-risk personnel, including animal caretakers and laboratory workers18,19. Refer to local authority regulations.
1. Fixation of brain tissue and sectioning
<ol>
	<li>Fix brain samples in an appropriate volume of 4% PFA in phosphate-buffered saline (PBS [pH 7.4]) for at least 48 h at 4 &#176;C (with an approximate tissue-to-fixative ratio of 1:10 [v/v]).</li>
	<li>Wash the tissue samples 3x in PBS for at least 30 min each wash and store them in 0.02% NaN3/PBS at 4 &#176;C until use.</li>
	<li>Section the tissue into 1 mm-thick sections using a vibratome (blade feed rate: 0.3&#8211;0.5 mm/s, amplitude: 1 mm, slice thickness: 1,000 &#181;m).</li>
	<li>To retain the correct slice sequence, store each tissue section separately in a well of a multiwell cell culture plate. Add 0.02% NaN3/PBS and store the tissue sections at 4 &#176;C until use.</li>
</ol>
2. Sample pretreatment with methanol
NOTE: Perform all incubation steps with gentle oscillation and, if not indicated otherwise, at room temperature. Protect the samples from light. The sample pretreatment serves the overall purpose of improving antibody diffusion and reducing tissue autofluorescence by exposure to MeOH and H2O2, respectively14.
<ol>
	<li>Prepare 20% (v/v), 40%, 60%, and 80% MeOH solutions in distilled water. For instance, for 20% MeOH, add 10 mL of 100% MeOH to 40 mL of distilled water in an appropriate sealable vessel and mix by inverting it.</li>
	<li>Transfer the samples to reasonably sized vessels (e.g., 5 mL reaction tubes). Take care to use materials chemically resistant to the reagents used in this protocol. For instance, note that while polypropylene is suitable, polystyrene is not. 
	NOTE: The volume specifications in this protocol refer to 5 mL reaction tubes. If a different vessel is used, adjust the volumes accordingly.</li>
	<li>Incubate the samples in 4 mL of each concentration of the prepared series of MeOH solutions in ascending order for 1 h each.</li>
	<li>Incubate the samples 2x for 1 h each in pure (100%) MeOH.</li>
	<li>Cool the samples to 4 &#176;C (e.g., in a laboratory-safe refrigerator).</li>
	<li>Prepare bleaching solution (5% H2O2 in MeOH) by, for instance, diluting a 30% H2O2 stock solution at 1:6 in pure (100%) MeOH, and chill it at 4 &#176;C.</li>
	<li>Remove the 100% MeOH from the refrigerated samples and add 4 mL of prechilled bleaching solution (5% H2O2 in MeOH). Incubate overnight at 4 &#176;C.</li>
	<li>Exchange the bleaching solution for 4 mL of 80% MeOH and incubate for 1 h. Continue using the prepared series of MeOH solutions in descending order for 1 h each until the samples have been incubated for 1 h in 4 mL of 20% MeOH.</li>
	<li>Wash the samples 1x for 1 h with 4 mL of PBS.</li>
</ol>
3. Immunostaining
NOTE: Perform all incubation steps with gentle oscillation and, if not indicated otherwise, at room temperature. Protect the samples from light. To prevent microbial growth, add NaN3 to a final concentration of 0.02% to the solutions in this section. Tissue samples are further permeabilized by treatment with nonionic detergents Triton X-100 and Tween 20. Normal serum is used to block unspecific antibody binding. Glycine and heparin are added to reduce the immunolabeling background14.
<ol>
	<li>Wash the samples 2x for 1 h each in 4 mL of 0.2% Triton X-100/PBS.</li>
	<li>Permeabilize the samples for 2 days at 37 &#176;C with 4 mL of 0.2% Triton X-100/20% DMSO/0.3 M glycine/PBS.</li>
	<li>Block the unspecific binding of antibodies by incubating the samples for 2 days at 37 &#176;C in 4 mL of 0.2% Triton X-100/10% DMSO/6% normal serum/PBS. 
	NOTE: Use normal serum from the same species the secondary antibody was raised in to achieve ideal blocking results.</li>
	<li>Incubate the samples in 2 mL of primary antibody solution (3% normal serum/5% DMSO/PTwH [PBS-Tween 20 with heparin] + primary antibody/antibodies) for 5 days at 37 &#176;C. Refresh the primary antibody solution&#160;after 2.5 days.
	<ol>
		<li>For PTwH, reconstitute the heparin sodium salt in distilled water to make a 10 mg/mL stock solution (store this solution, aliquoted, at 4 &#176;C). Add the stock solution to 0.2% Tween 20/PBS to a final concentration of 10 &#181;g/mL. 
		NOTE: Choosing the correct antibody dilution may require optimization. Generally, standard immunohistochemistry concentrations are a good starting point.</li>
	</ol>
	</li>
	<li>Wash the samples for 1 day in 4 mL of PTwH, exchanging the wash buffer at least 4x&#8211;5x during the course of the day and leaving the final wash on overnight.</li>
	<li>Incubate the samples in 2 mL of secondary antibody solution (3% normal serum/PTwH + secondary antibody/antibodies) for 5 days at 37 &#176;C. Refresh the secondary antibody solution after 2.5 days.
	<ol>
		<li>Dilute the secondary antibody/antibodies at 1:500 in secondary antibody solution (i.e., 4 &#181;L in 2 mL).</li>
	</ol>
	</li>
	<li>Wash the samples for 1 day as described in step 3.5, leaving the final wash on overnight.</li>
</ol>
4. Nuclear staining
NOTE: Perform all incubation steps with gentle oscillation and, if not indicated otherwise, at room temperature. Protect the samples from light. If no nuclear staining is required or the excitation wavelength/emission spectrum of TO-PRO-3 is required for the excitation or detection of another fluorophore, skip this step.
<ol>
	<li>Dilute the nucleic acid stain TO-PRO-3 at 1:1,000 in PTwH and incubate the samples in 4 mL of nuclear staining solution for 5 h.</li>
	<li>Wash the samples for 1 day as described in step 3.5, leaving the final wash on overnight. 
	NOTE: Following the washes, the samples can be stored in PBS at 4 &#176;C until optical clearing.</li>
</ol>
5. Tissue clearing
NOTE: Perform all incubation steps with gentle oscillation and, if not indicated otherwise, at room temperature. Protect the samples from light. The tissue samples are dehydrated in a graded series of TBA solutions. As immunostaining requires aqueous solutions, all staining procedures have to be finished prior to tissue clearing. Optical clearance and refractive index matching are achieved by treatment with a mixture of BA, BB, and DPE. The clearing solution is supplemented with DL-&#945;-tocopherol as an antioxidant13.
<ol>
	<li>Prepare 30% (v/v), 50%, 70%, 80%, 90%, and 96% TBA solutions in distilled water. For instance, for 30% TBA, add 15 mL of 100% TBA to 35 mL of distilled water in an appropriate sealable vessel and mix by inverting. 
	NOTE: TBA has a melting point of 25&#8211;26 &#176;C; thus, it tends to be solid at room temperature. In order to prepare TBA solutions, heat the well-sealed bottle at 37 &#176;C in an incubator or water bath.</li>
	<li>Dehydrate the samples with 4 mL of each concentration of the prepared series of TBA solutions in ascending order for 2 h each. Leave the 96% TBA on overnight.</li>
	<li>Dehydrate the samples further in pure (100%) TBA for 2 h.</li>
	<li>Prepare clearing solution BABB-D15. 
	NOTE: BABB-D15 is a combination of BA and BB (BABB) which is mixed with DPE at a ratio of x:1, where x is specified in the solution&#8217;s name, in this case 15.
	<ol>
		<li>For BABB, mix one-part BA with two parts BB.</li>
		<li>Mix BABB and DPE at a ratio of 15:1.</li>
		<li>Add 0.4 vol% DL-&#945;-tocopherol (vitamin E). 
		NOTE: For example, for 20 mL of BABB-D15, mix 6.25 mL of BA with 12.5 mL of BB. Add 1.25 mL of DPE and supplement it with 0.08 mL of DL-&#945;-tocopherol.</li>
	</ol>
	</li>
	<li>Clear the samples in clearing solution until they are optically transparent (2&#8211;6 h).</li>
	<li>The samples can be stored at 4 &#176;C in BABB-D15, protected from light, until mounting and imaging.</li>
</ol>
6. Sample mounting
<ol>
	<li>Using a 3D printer, print the&#160;imaging chamber and the lid (material: copolyester [CPE], nozzle: 0.25 mm, layer height: 0.06 mm, wall thickness: 0.88 mm, wall count: 4, infill: 100%, no support structure; the corresponding .STL file can be found in the <a href="https://www.jove.com/files/ftp_upload/59402/3D_ImagingChamber.stl" target="_blank">Supplementary Materials</a> of this protocol).</li>
	<li>Assemble the imaging chamber (Figure 2).
	<ol>
		<li>Mount a round coverslip (diameter: 30 mm) on the imaging chamber with RTV-1 (one-component room-temperature-vulcanizing) silicone rubber. Remove the excess silicone rubber with a water-wetted cotton swab and cure overnight.</li>
		<li>Mount a round coverslip (diameter: 22 mm) on the lid with RTV-1 silicone rubber. Remove the&#160;excess silicone rubber&#160;with a water-wetted cotton swab and cure overnight.</li>
	</ol>
	</li>
	<li>Place the sample in an imaging chamber, add a small volume of BABB-D15, and insert the lid. Fill the chamber up with BABB-D15 through the inlet, using a hypodermic needle (27 G x 3/4 inch [0.40 mm x 20 mm]).</li>
	<li>Plug the inlet and seal the imaging chamber with RTV-1 silicone rubber. Cure overnight in the dark.</li>
</ol>
7. Imaging and image processing
<ol>
	<li>Set up the image acquisition by selecting the respective laser lines to match the fluorophores used. Adjust the detection ranges of each detector to prevent signal overlap between channels. 
	NOTE: Exemplary detection ranges for Alexa Fluor 488, Alexa Fluor 568, and TO-PRO-3 are 500&#8211;550 nm, 590&#8211;620 nm, and 645&#8211;700 nm, respectively.</li>
	<li>Choose the acquisition parameters, define the upper and lower border of the z-stack, and acquire the image stack. 
	NOTE: Exemplary acquisition parameters are a sequential scan with a pixel size of 60-90 nm, a z-step size of 0.5 &#181;m, a line average of 1, a scan speed of 400 Hz, and a pinhole size of 1 Airy unit.</li>
	<li>Process the image stack using appropriate image analysis software (e.g., Fiji) to generate 3D projections or perform in-depth analyses. 
	NOTE: Because of the large size of the acquired image files, the use of a workstation is usually necessary.
	<ol>
		<li>Open the acquisition or image file(s) in Fiji (File | Open | Select files).
		<ol>
			<li>If using, for instance, .LIF files, select or deselect the desired options in the Bio-Formats dialog window. View stack with Hyperstack. Other than that, no specific selections or ticks are necessary. Press OK.</li>
			<li>If the file contains multiple image stacks, select the ones to be analyzed and confirm by pressing OK.</li>
		</ol>
		</li>
		<li>Perform bleach correction by splitting the merged image into individual channels (Image | Color | Channels Tool, then select More | Split Channels). For each channel, select bleach correction (Image | Adjust | Bleach Correction) and choose Simple Ratio (background intensity: 0.0). 
		NOTE: In some cases, for instance, when there is no linear decay of the signal or the signal is too weak overall, Simple Ratio may fail. Alternatively, try Exponential Fit or skip the bleach correction.</li>
		<li>Adjust the brightness and contrast for each channel using the sliders (Image | Adjust | Brightness | Contrast).</li>
		<li>Merge the channels (Image | Color | Merge Channels), make a composite (Image | Color | Channels Tool, then select More | Make Composite), and convert it to RGB format (Image | Color | Channels Tool, then select More | Convert to RGB).</li>
		<li>If necessary, resize the image stack to reduce the computation time and file size (Image | Adjust | Size, both options ticked plus bilinear interpolation).</li>
		<li>Generate a 3D projection (Image | Stacks | 3D Project). Choose Brightest Point as 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.</li>
		<li>If necessary, readjust the contrast and brightness by converting the 3D projection back to 8-bit format (Image | Type | 8-bit). Use the respective sliders (Image | Adjust | Brightness | Contrast) and reconvert the image stack to RGB format as described in step 7.3.4.</li>
		<li>Save the 3D projection as .TIF file (image file format) and .AVI file (video file format).</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The resurgence and further development of tissue clearing techniques in recent years2,3,4,5,6,7,8,9,10,11,12,13,14 have opened up many new possibilities to obtain high-volume image stacks of organ tissue. This provided an unparalleled and powerful tool to study, among many other topics, virus infection. The subsequent 3D reconstruction of these image stacks enables sophisticated assertions on, for instance, virus tropism, abundance, and the time course of infection. This protocol describes the immunolabeling-assisted visualization of RABV infection in solvent-cleared brain tissue.
There are several critical steps in the preparation and acquisition of the image stacks of immunolabeled, solvent-cleared tissue. Prolonged exposure to PFA can mask epitopes and, thus, result in decreased antigenicity20,21,22. It is, therefore, important to limit the fixation times to the necessary minimum and transfer the samples to an appropriate solution (e.g., PBS supplemented with 0.02% NaN3 for long-term storage). However, all representative images and projections provided here have been acquired from archived brain samples, some of which had been stored in PFA for weeks, highlighting the applicability of the technique to organ material that has been exposed to PFA for extended periods of time. Similar results have been shown for human brain samples13. Another, if not the most, critical step is the antibody incubation. Antibody concentrations have to be chosen carefully. While in most cases, standard IHC concentrations are a good starting point for deep-tissue antibody labeling, some antigens may require additional optimization of the antibody concentration. This protocol demonstrates that both RABV N and P are readily detectable by the used antibodies. While MeOH pretreatment usually improves immunolabeling, some antigens are incompatible with this treatment. For those, Renier and colleagues14 provided an alternative MeOH-free sample pretreatment. Analysis of the acquired image stacks requires adequate computational power. Because of the large file sizes of up to several gigabytes per stack, powerful computers are needed to process the images. Postprocessing often includes the subtraction of background noise and a bleach correction to compensate for acquisition-associated bleaching effects. When measuring distances, it has to be kept in mind that, as a side effect, the tissue is shrinking during the clearing process.
In comparison to other microscopy platforms, like light sheet fluorescence microscopy (LSFM) or two-photon laser scanning microscopy (2PLSM), CLSM has the most limited working distance. Therefore, it is necessary to preslice organs to 1 mm in thickness for imaging. Additionally, CLSM has the slowest acquisition speed because of its high imaging resolution. The use of a light sheet microscope would allow for faster imaging of larger volumes up to whole-brain imaging, while simultaneously sacrificing image resolution. Another limitation is the inherent increase of tissue autofluorescence with decreasing wavelength. This renders the use of fluorophores and dyes excited by the laser line at 405 nm, including Hoechst dyes, impractical to impossible.
With respect to other tissue clearing techniques, the hybrid of iDISCO14 and uDISCO13 combines the most positive attributes: it is highly compatible with immunostaining, highly versatile, comparatively fast&#160;and inexpensive, it has excellent clearing capabilities, and it is feasible with a standard confocal microscope. Moreover, this protocol is not restricted to the immunostaining and clearing of brain tissue but can also be applied to a variety of other soft tissues and pathogens, both neuroinvasive and non-neuroinvasive. In conclusion, the protocol described here represents a pipeline for high-resolution 3D imaging of RABV-infected brain tissue. The 3D reconstructions of infected brain tissue can be used to answer a variety of questions concerning RABV disease progression, pathogenesis, and neuroinvasion.

Conventional histology techniques mostly rely on thin sections of organ tissues, which can inherently provide only 2D insights into a complex 3D environment. Although feasible in principle, 3D reconstruction from serial thin sections requires demanding technical pipelines for both slicing and subsequent in silico alignment of the acquired images1. Moreover, seamless reconstruction of z-volumes after microtome slicing is critical as both mechanical and computational artifacts can remain because of suboptimal image registration caused by nonoverlapping image planes, staining variations, and physical destruction of tissue by, for instance, the microtome blade. In contrast, pure optical slicing of intact thick tissue samples allows the acquisition of overlapping image planes (oversampling) and, thereby, facilitates 3D reconstruction. This, in turn, is highly beneficial for the analysis of infection processes in complex cell populations (e.g., neuronal networks in the context of the surrounding glial and immune cells). However, inherent obstacles of thick tissue sections include light scattering and limited antibody penetration into the tissue. In recent years, a variety of techniques has been developed and optimized to overcome these issues2,3,4,5,6,7,8,9,10,11,12,13. Essentially, target tissues are turned optically transparent by treatment with either aqueous2,3,4,5,6,7,8,9 or organic solvent-based10,11,12,13 solutions. The introduction of 3DISCO (3D imaging of solvent-cleared organs)11,12 and its successor uDISCO (ultimate 3D imaging of solvent-cleared organs)13 provided a relatively fast, simple, and inexpensive tool with excellent clearing capabilities. The main constituents of the clearing protocol are the organic solvents tert-butanol (TBA), benzyl alcohol (BA), benzyl benzoate (BB), and diphenyl ether (DPE). The development and addition of iDISCO (immunolabeling-enabled 3D imaging of solvent-cleared organs)14, a compatible immunostaining protocol, constituted another advantage over existing methods and enabled the deep-tissue labeling of antigens of interest, as well as the long-term storage of immunostained samples. Thus, the combination of iDISCO14 and uDISCO13 allows for the high-resolution imaging of antibody-labeled proteins in large tissue sections (up to 1 mm) using conventional CLSM.
The preservation of an organ&#8217;s complex structure in all three dimensions is particularly important for brain tissue. Neurons comprise a very heterogeneous cellular subpopulation with highly diverse 3D morphologies based on their neurite projections (reviewed by Masland15). Furthermore, the brain consists of a number of compartments and subcompartments, each composed of different cellular subpopulations and ratios thereof, including glial cells and neurons (reviewed by von Bartheld et al.16). As a neurotropic virus, the rabies virus (RABV, reviewed by Fooks et al.17) primarily infects neurons, using their transport machinery to travel in retrograde direction along axons from the primary site of infection to the central nervous system (CNS). The protocol described here (Figure 1A) allows for the immunostaining-assisted detection and visualization of RABV and RABV-infected cells in large, coherent image stacks obtained from infected brain tissue. This enables an unbiased, 3D high-resolution assessment of the infection environment. It is applicable to brain tissue from a variety of species, can be performed immediately after fixation or after the long-term storage of samples in paraformaldehyde (PFA), and allows the storage and reimaging of stained and cleared samples for months.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Benzyl alcohol</td><td>Alfa Aesar</td><td>41218</td><td>Clearing reagent</td></tr><tr><td>Benzyl benzoate</td><td>Sigma-Aldrich</td><td>BB6630-500ML</td><td>Clearing reagent</td></tr><tr><td>Dimethyl sulfoxide</td><td>Carl Roth</td><td>4720.2</td><td>Various buffers</td></tr><tr><td>Diphenyl ether</td><td>Sigma-Aldrich</td><td>240834-100G</td><td>Clearing reagent</td></tr><tr><td>DL-&amp;alpha;-Tocopherol</td><td>Alfa Aesar</td><td>A17039</td><td>Antioxidant</td></tr><tr><td>Donkey serum</td><td>Bio-Rad</td><td>C06SBZ</td><td>Blocking reagent</td></tr><tr><td>Glycine</td><td>Carl Roth</td><td>3908.2</td><td>Background reduction</td></tr><tr><td>Goat serum</td><td>Merck</td><td>S26-100ML</td><td>Blocking reagent</td></tr><tr><td>Heparin sodium salt</td><td>Carl Roth</td><td>7692.1</td><td>Background reduction</td></tr><tr><td>Hydrogen peroxide solution (30 %)</td><td>Carl Roth</td><td>8070.2</td><td>Sample bleaching</td></tr><tr><td>Methanol</td><td>Carl Roth</td><td>4627.4</td><td>Sample pretreatment</td></tr><tr><td>Paraformaldehyde</td><td>Carl Roth</td><td>0335.3</td><td>Crystalline powder to make fixative solution</td></tr><tr><td>Sodium azide</td><td>Carl Roth</td><td>K305.1</td><td>Prevention of microbial growth in stock solutions</td></tr><tr><td>tert-Butanol</td><td>Alfa Aesar</td><td>33278</td><td>Sample dehydration for tissue clearing</td></tr><tr><td>TO-PRO-3</td><td>Thermo Fisher</td><td>T3605</td><td>Nucleic acid stain</td></tr><tr><td>Triton X-100</td><td>Carl Roth</td><td>3051.2</td><td>Detergent</td></tr><tr><td>Tween 20</td><td>AppliChem</td><td>A4974,0500</td><td>Detergent</td></tr><tr><td>&lt;strong&gt;Miscellaneous&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>5 mL reaction tubes</td><td>Eppendorf</td><td>0030119401</td><td>Sample tubes</td></tr><tr><td>Coverslip, circular (diameter: 22 mm)</td><td>Marienfeld</td><td>0111620</td><td>Part of imaging chamber</td></tr><tr><td>Coverslip, circular (diameter: 30 mm)</td><td>Marienfeld</td><td>0111700</td><td>Part of imaging chamber</td></tr><tr><td>Hypodermic needle (27 G x &amp;frac34;&amp;rdquo; [0.40 mm x 20 mm])</td><td>B. Braun</td><td>4657705</td><td>Filling of the imaging chamber with clearing solution</td></tr><tr><td>RTV-1 silicone rubber</td><td>Wacker</td><td>Elastosil E43</td><td>Adhesive for the assembly of the imaging chamber</td></tr><tr><td>Ultimaker CPE 2.85 mm transparent</td><td>Ultimaker</td><td>8718836374869</td><td>Copolyester filament for 3D printer to print parts of the imaging chamber</td></tr><tr><td>&lt;strong&gt;Technical equipment and software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>3D printer</td><td>Ultimaker</td><td>Ultimaker 2+</td><td>Printing of imaging chamber</td></tr><tr><td>Automated water immersion system</td><td>Leica</td><td>15640019</td><td>Software-controlled water pump</td></tr><tr><td>Benchtop orbital shaker</td><td>Elmi</td><td>DOS-20M</td><td>Sample incubation at room temperature (~ 150 rpm)</td></tr><tr><td>Benchtop orbital shaker, heated</td><td>New Brunswick Scientific</td><td>G24 Environmental Shaker</td><td>Sample incubation at 37 &amp;deg;C (~ 150 rpm)</td></tr><tr><td>Confocal laser scanning microscope</td><td>Leica</td><td>DMI 6000 TCS SP5</td><td>Inverted confocal microscope for sample imaging</td></tr><tr><td>Fiji</td><td>NIH (ImageJ)</td><td>open source software (v1.52h)</td><td>Image processing package based on ImageJ</td></tr><tr><td>Long working distance water immersion objective</td><td>Leica</td><td>15506360</td><td>HC PL APO 40x/1.10 W motCORR CS2</td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1200S</td><td>Sample slicing</td></tr><tr><td>Workstation</td><td>Dell</td><td>Precision 7920</td><td>CPU: Intel Xeon Gold 5118&lt;br/&gt; GPU: Nvidia Quadro P5000&lt;br/&gt; RAM: 128 GB 2666 MHz DDR4&lt;br/&gt; SSD: 2 TB</td></tr><tr><td>&lt;strong&gt;Primary antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Goat anti-RABV N</td><td>Friedrich-Loeffler-Institut</td><td></td><td>Monospecific polyclonal goat anti-RABV N serum, generated by goat immunization with baculovirus-expressed and His-tag-purified RABV nucleoprotein N&lt;br/&gt; Dilution: 1:400</td></tr><tr><td>Rabbit anti-GFAP</td><td>Dako</td><td>Z0334</td><td>Polyclonal antibody (RRID:AB_10013382)&lt;br/&gt; Dilution: 1:100</td></tr><tr><td>Rabbit anti-MAP2</td><td>Abcam</td><td>ab32454</td><td>Polyclonal antibody (RRID:AB_776174)&lt;br/&gt; Dilution: 1:250</td></tr><tr><td>Rabbit anti-RABV P 160-5</td><td>Friedrich-Loeffler-Institut</td><td></td><td>Monospecific polyclonal rabbit anti-RABV P serum, generated by rabbit immunization with baculovirus-expressed and His-tag-purified RABV phosphoprotein P (see reference 23: Orbanz et al., 2010)&lt;br/&gt; Dilution: 1:1,000</td></tr><tr><td>&lt;strong&gt;Secondary antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Donkey anti-goat IgG</td><td>Thermo Fisher Scientific</td><td>depending on conjugated fluorophore</td><td>Highly cross-absorbed&lt;br/&gt; Dilution: 1:500</td></tr><tr><td>Donkey anti-mouse IgG</td><td>Thermo Fisher Scientific</td><td>depending on conjugated fluorophore</td><td>Highly cross-absorbed&lt;br/&gt; Dilution: 1:500</td></tr><tr><td>Donkey anti-rabbit IgG</td><td>Thermo Fisher Scientific</td><td>depending on conjugated fluorophore</td><td>Highly cross-absorbed&lt;br/&gt; Dilution: 1:500</td></tr><tr><td>Goat anti-mouse IgG</td><td>Thermo Fisher Scientific</td><td>depending on conjugated fluorophore</td><td>Highly cross-absorbed&lt;br/&gt; Dilution: 1:500</td></tr><tr><td>Goat anti-rabbit IgG</td><td>Thermo Fisher Scientific</td><td>depending on conjugated fluorophore</td><td>Highly cross-absorbed&lt;br/&gt; Dilution: 1:500</td></tr></tbody></table>

high-resolution 3d imaging of rabies virus infection in solvent-cleared brain tissue

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample&#8217;s refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

The combination of iDISCO14 and uDISCO13 coupled with high-resolution CLSM provides deep insights into the spatiotemporal resolution and plasticity of RABV infection of brain tissue and the surrounding cellular context.
Using immunostaining of RABV phosphoprotein (P), complex layers of infected neuronal cells can be visualized in thick sections of mouse brains (Figure 3). Subsequently, seamless 3D projections of the acquired image stacks can be reconstructed (Figure 3A, B, right panel; Animated Figure 1). Care has to be taken when using primary antibodies from and secondary antibodies against the species the organ material originates from. The use of anti-mouse IgG antibodies on mouse brain tissue resulted in a distinct staining of the vascular system (Figure 3B, left panel). Because of the high resolution the image stacks are acquired with, infection can be assessed up to a single-cell level (Figure 4), allowing assertions on, for instance, the abundance and distribution of antigen within the cell (Figure 4C).
Aside from mouse brain tissue, the protocol can also be applied to brain tissue from other animal species (e.g., ferrets) (Figure 5; Animated Figure 2). Sections taken from different compartments of an infected ferret brain revealed a varying degree of RABV infection (Figure 5A-D).
As the brain comprises many different cellular subpopulations, differentiation between these populations is vital. Using antibodies directed against cell markers, assessment of the cellular identity of infected and neighboring cells is possible. For instance, astrocytes can be differentiated via the expression of glial fibrillary acidic protein (GFAP) (Figure 6A,C; Animated Figure 3), while neurites can be specifically stained for microtubule-associated protein 2 (MAP2) (Figure 6B,D; Animated Figure 4). Simultaneously, viral proteins, in this case, RABV nucleoprotein (N), can be costained to assess the relation between infected cells and the highlighted cellular subpopulation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59402/59402fig1.jpg" /> 
Figure 1: Basic principle and workflow of the protocol. (A) Graphical representation of the workflow based on the protocols from Renier et al.14 and Pan et al.13. (B) Two exemplary ferret brain slices, one before (left panel) and one after treatment with organic solvents (right panel). Clearing turns the tissue optically transparent as observable by the then readable text. The cleared brain slice is embedded in a 3D-printed imaging chamber (right panel). <a href="https://www.jove.com/files/ftp_upload/59402/59402fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59402/59402fig2.jpg" /> 
Figure 2: Technical illustration of the 3D-printed imaging chamber. (A) Exploded view drawing of the imaging chamber. The dot-dashed lines highlight components that have to be mounted on each other using RTV-1 silicone rubber. The dashed lines represent instructions for the subsequent assembly of the chamber. (B) CAD (computer-aided design) file of the imaging chamber. The corresponding .STL file to print the imaging chamber can be found in the <a href="https://www.jove.com/files/ftp_upload/59402/3D_ImagingChamber.stl" target="_blank">Supplementary Materials</a>. (C) Fully assembled imaging chamber. <a href="https://www.jove.com/files/ftp_upload/59402/59402fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59402/59402fig3.jpg" /> 
Figure 3: Deep-tissue imaging of RABV-infected mouse brain tissue. (A and B) Mice were infected with a recombinant vulpine street virus. RABV P staining (green) reveals&#160;infected neuronal layers with large, entangled neurite projections inside the cleared brain tissue. Nuclei were counterstained with TO-PRO-3 (blue). (B) The use of fluorophore-labeled anti-mouse IgG secondary antibodies (red) on mouse tissue results&#160;in a distinct labeling of the vascular system. The 3D reconstruction of the acquired image stacks enables observation from different viewing angles (A and B, right panels). Scale bars = 60 &#181;m. <a href="https://www.jove.com/files/ftp_upload/59402/59402fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59402/59402fig4.jpg" /> 
Figure 4: High-resolution image acquisition enables complex assessments up to a single-cell level. The brain was dissected from an SAD L16-infected mouse. (A) RABV P staining (green) highlights an individually infected neuron. (B and C) Detail images and projections demonstrate that in-depth analyses of antigen abundance, distribution, and localization can be performed. Nuclei were counterstained with TO-PRO-3 (blue). Scale bars = 20 &#181;m (A),&#160;5 &#181;m (C). <a href="https://www.jove.com/files/ftp_upload/59402/59402fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59402/59402fig5.jpg" /> 
Figure 5: Deep-tissue imaging of RABV-infected ferret brain tissue. Ferrets were infected with canine street RABV. (A&#8211;D) Slices from specified areas of the brain were taken, immunostained for RABV P (green), and optically cleared. Projections demonstrate that the infected cells in different parts of the brain differ in amount and morphology. Furthermore, they highlight the applicability of the protocol to tissues other than mouse-derived. Nuclei were counterstained with TO-PRO-3 (blue). Scale bars = 60 &#181;m. <a href="https://www.jove.com/files/ftp_upload/59402/59402fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59402/59402fig6.jpg" /> 
Figure 6: Multicolor immunofluorescence allows the costaining of cellular markers. Brain tissue slices from ferrets infected with canine street RABV were used and coimmunostained for RABV N (red) and either (A and C) GFAP (green) or (B and D) MAP2 (green). Whereas GFAP is an astrocyte marker, MAP2 specifically highlights neurites. Nuclei were counterstained with TO-PRO-3 (blue). (C and D) At the bottom, single slice extractions of the enumerated detail views from the merged projections are depicted. Scale bars = 15 &#181;m (A and B)&#160;10 &#181;m (C and D). <a href="https://www.jove.com/files/ftp_upload/59402/59402fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Animated Figure 1" src="/files/ftp_upload/59402/59402anifig1.jpg" /> 
Animated Figure 1: 3D reconstructions and detail projections of image stacks from RABV-infected mouse brains. The projections were generated from the image stacks described in Figure 3. (A and C) Animations of the entire respective z-stacks. (B) A detailed projection of a 3D reconstruction of a part of the z-stack of the areas highlighted at the beginning of the video. (D) A detailed projection of a tomogram of a part of the z-stack of the areas highlighted at the beginning of the video. Green = RABV P; red = mouse IgG; blue = nuclei. <a href="https://www.jove.com/files/ftp_upload/59402/Animation1_NeuronsCombinedAnimation_compressed_remuxed.mp4" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Animated Figure 2" src="/files/ftp_upload/59402/59402anifig2.jpg" /> 
Animated Figure 2: 3D reconstructions of image stacks acquired from an RABV-infected ferret brain. The projections were generated from the image stacks described in Figure 5. (A&#8211;D) The annotations refer to the same figure and describe the respective areas of the brain the slices were taken from. Green = RABV P; blue = nuclei. <a href="https://www.jove.com/files/ftp_upload/59402/Animation2_CombinedAnimationFerretBrain_compressed_remuxed.mp4" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Animated Figure 3" src="/files/ftp_upload/59402/59402anifig3.jpg" /> 
Animated Figure 3: Gradual projection of the different channels of a 3D reconstruction of an RABV-infected ferret brain costained with astrocyte marker GFAP. Projections were generated from the image stack described in Figure 6A. The gradual addition of channels starts with RABV N (red), after which follow the cell nuclei (blue) and, finally, GFAP (green), while the subtraction first removes RABV N, then the cell nuclei, and eventually GFAP. <a href="https://www.jove.com/files/ftp_upload/59402/Animation3_GradualChannelProjectionGFAP_compressed_remuxed.mp4" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Animated Figure 4" src="/files/ftp_upload/59402/59402anifig4.jpg" /> 
Animated Figure 4: Gradual projection of the different channels of a 3D reconstruction of an RABV-infected ferret brain costained with neuronal marker MAP2. Projections were generated from the image stack described in Figure 6B. The gradual addition of channels starts with RABV N (red), after which follow the cell nuclei (blue) and, finally, MAP2 (green), while the subtraction first removes RABV N, then the cell nuclei, and eventually MAP2. <a href="https://www.jove.com/files/ftp_upload/59402/Animation4_GradualChannelProjectionMAP2_compressed_remuxed.mp4" target="_blank">Please click here to view this video. (Right-click to download.)</a>

Watch this Scientific Journal Video about High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue at JoVE.com

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...

high-resolution-3d-imaging-rabies-virus-infection-solvent-cleared

Universidad Científica del Sur

Research

JoVE Journal

Neuroscience

10.9K Views.  Federal Research Institute for Animal Health. Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

Video: High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma establishes a lifelong persistent infection in the brain. While this brain infection is asymptomatic in most immunocompetent people, in the developing fetus or immunocompromised individuals such as acquired immune deficiency syndrome (AIDS) patients, this predilection for and persistence in the brain can lead to devastating neurologic disease. Thus, it is clear that the brain-Toxoplasma interaction is critical to the symptomatic disease produced by Toxoplasma, yet we have little understanding of the cellular or molecular interaction between cells of the central nervous system (CNS) and the parasite. In the mouse model of CNS toxoplasmosis it has been known for over 30 years that neurons are the cells in which the parasite persists, but little information is available about which part of the neuron is generally infected (soma, dendrite, axon) and if this cellular relationship changes between strains. In part, this lack is secondary to the difficulty of imaging and visualizing whole infected neurons from an animal. Such images would typically require serial sectioning and stitching of tissue imaged by electron microscopy or confocal microscopy after immunostaining. By combining several techniques, the method described here enables the use of thick sections (160 &#181;m) to identify and image whole cells that contain cysts, allowing three-dimensional visualization and analysis of individual, chronically infected neurons without the need for immunostaining, electron microscopy, or serial sectioning and stitching. Using this technique, we can begin to understand the cellular relationship between the parasite and the infected neuron.

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Using this protocol, we were able to image 160 &#181;m thick brain sections from mice infected with the parasite Toxoplasma gondii, which enables visualization and analysis of the spatial relationship between the encysting parasite and the infected neuron.

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma ...

Enhanced Rabies Surveillance Using a Direct Rapid Immunohistochemical Test

Laboratory-based surveillance is integral for rabies prevention, control and management efforts. While the DFA is the gold standard for rabies diagnosis, there is a need to validate additional diagnostic techniques to improve rabies surveillance, particularly in developing countries. Here, we present a standard protocol for the DRIT as an alternative, laboratory or field-based testing option that uses light microscopy as compared to the DFA. Touch impressions of brain tissue collected from suspect animals are fixed in 10% buffered formalin. The DRIT uses rabies virus-specific monoclonal or polyclonal antibodies (conjugated to biotin), a streptavidin-peroxidase enzyme, and a chromogen reporter (such as acetyl 3-amino-9-ethylcarbazole) to detect viral inclusions within infected tissue. In approximately 1 h, a brain tissue sample can be tested and interpreted by the DRIT. Evaluation of suspect animal brains tested from a variety of species in North America, Asia, Africa, and Europe have illustrated high sensitivity and specificity by the DRIT approaching 100% with results compared to DFA. Since 2005, the United States Department of Agriculture&#8217;s Wildlife Services (USDA WS) program has conducted large-scale enhanced rabies surveillance efforts using the DRIT to test &#62;94,000 samples collected from wildlife in strategic rabies management areas. The DRIT provides a powerful, economical tool for rabies diagnosis that can be used by laboratorians and field biologists to improve current rabies surveillance, prevention and control programs globally.

The direct rapid immunohistochemical test (DRIT) offers a World Organization for Animal Health and World Health Organization (OIE/WHO) recognized alternative to the direct fluorescent antibody (DFA) test for rabies diagnosis. This test allows for field-based applications that can be performed in approximately 1 h upon brain impressions using light microscopy.

Laboratory-based surveillance is integral for rabies prevention, control and management efforts. While the DFA is the gold standard for rabies diagnosis, ...

Immunology and Infection

Quantitation of Rabies Virus in Various Bovine Brain Structures

Bovine paralytic rabies (BPR) is a form of viral encephalitis that is of substantial economic importance throughout Latin America, where it poses a major zoonotic risk. Here, our objective was to utilize a laboratory protocol to determine the relative copy number of the rabies virus (RABV) genome in different bovine brain anatomical structures using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). qRT-PCR quantifies the specific number of gene copies present in a sample based on fluorescence emitted after amplification that is directly proportional to the amount of target nucleic acid present in the sample. This method is advantageous owing to its short duration, reduced risk of contamination, and potential to detect viral nucleic acids in different samples more easily compared to other techniques. The brains of six rabid animals were divided into six anatomical structures, namely the Ammon&#8217;s horn, cerebellum, cortex, medulla, pons, and thalamus. All brains were identified as positive for RABV antigens based on a direct immunofluorescence test. The same anatomical structures from the brains of four RABV-negative bovines were also assessed. RNA was extracted from each structure and used for qRT-PCR. An assay was performed to determine the copy numbers of RABV genes using an in vitro transcribed nucleoprotein gene. The standard curve used to quantify viral RNA exhibited an efficiency of 100% and linearity of 0.99. Analysis revealed that the cortex, medulla, and thalamus were the ideal CNS portions for use in RABV detection, based on the observation that these structures possessed the highest levels of RABV. The test specificity was 100%. All samples were positive, no false positives were detected. This method can be used to detect RABV in samples that contain low levels of RABV during diagnosis of BPR.

This protocol presents a qRT-PCR-based approach for determining the rabies virus nucleoprotein (N) gene copy number within various bovine brain anatomical structures using in vitro transcription.

Bovine paralytic rabies (BPR) is a form of viral encephalitis that is of substantial economic importance throughout Latin America, where it poses a major ...

A Rat Model of EcoHIV Brain Infection

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. Establishment of the EcoHIV infected rat model for studies of drug abuse and neurocognitive disorders, would be beneficial in the study of neuroHIV and HIV-1 associated neurocognitive disorders (HAND). In the present study, we demonstrate the successful creation of a rat model of active HIV infection using chimeric HIV (EcoHIV). First, the lentiviral construct of EcoHIV was packaged in cultured 293 FT cells for 48 hours. Then, the conditional medium was concentrated and titered. Next, we performed bilateral stereotaxic injections of the EcoHIV-EGFP into F344/N rat brain tissue. One week after infection, EGFP fluorescence signals were detected in the infected brain tissue, indicating that EcoHIV successfully induces an active HIV infection in rats. In addition, immunostaining for the microglial cell marker, Iba1, was performed. The results indicated that microglia were the predominant cell type harboring EcoHIV. Furthermore, EcoHIV rats exhibited alterations in temporal processing, a potential underlying neurobehavioral mechanism of HAND as well as synaptic dysfunction eight weeks after infection. Collectively, the present study extends the EcoHIV model of HIV-1 infection to the rat offering a valuable biological system to study HIV-1 viral reservoirs in the brain as well as HAND and associated comorbidities such as drug abuse.

Here, we present a protocol to establish a new rat model of active HIV infection using chimeric HIV (EcoHIV), which is critical for enhancing our understanding of HIV-1 viral reservoirs in the brain and offering a system to study HIV-associated neurocognitive disorders and associated comorbidities (i.e., drug abuse).

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. ...

A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging from neural circuits to subcellular neuronal structures are visualized in mouse brain slices optically cleared with ScaleSF. This clearing method is a modified version of ScaleS and is a hydrophilic tissue clearing method for tissue slices that achieves potent clearing capability as well as a high-level of preservation of fluorescence signals and structural integrity. A customizable three dimensional (3D)-printed imaging chamber is designed for reliable mounting of cleared brain tissues. Mouse brains injected with an adeno-associated virus vector carrying enhanced green fluorescent protein gene were fixed with 4% paraformaldehyde and cut into slices of 1-mm thickness with a vibrating tissue slicer. The brain slices were cleared by following the clearing protocol, which include sequential incubations in three solutions, namely, ScaleS0 solution, phosphate buffer saline (&#8211;), and ScaleS4 solution, for a total of 10.5&#8211;14.5 h. The cleared brain slices were mounted on the imaging chamber and embedded in 1.5% agarose gel dissolved in ScaleS4D25(0) solution. The 3D image acquisition of the slices was carried out using a confocal laser scanning microscope equipped with a multi-immersion objective lens of a long working distance. Beginning with mesoscopic neuronal imaging, we succeeded in visualizing fine subcellular neuronal structures, such as dendritic spines and axonal boutons, in the optically cleared brain slices. This protocol would facilitate understanding of neuronal structures from circuit to subcellular component scales.

The protocol provides a detailed method of neuronal imaging in brain slice using a tissue clearing method, ScaleSF. The protocol includes brain tissue preparation, tissue clarification, handling of cleared slices and confocal laser scanning microscopy imaging of neuronal structures from mesoscopic to microscopic levels.

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging ...

Postmortem Diagnosis of Rabies in Animals by the Updated, Multiplexed LN34 Real-Time Reverse Transcription-Polymerase Chain Reaction Assay

Rabies is a fatal zoonotic disease caused by Lyssavirus rabies (RABV) and related negative strand RNA viruses from the Lyssavirus genus (family Rhabdoviridae). The LN34 assay targets the highly conserved leader region and nucleoprotein gene of the lyssavirus genome and utilizes degenerate primers and a TaqMan probe containing locked nucleotides to detect RNA across the diverse Lyssavirus genus. A negative finding for rabies should be made only if a full cross-section of the brain stem and three lobes of the cerebellum are examined; however, identification of lyssavirus RNA in any tissue is diagnostic of rabies infection. Tissue is collected and homogenized in TRIzol reagent, which also inactivates the virus. RNA extraction is performed using a spin column-based commercial extraction kit. Master mixes are prepared in a clean space and aliquoted into a 96 well plate before adding sample RNA. In clinical settings, each sample is tested by real-time RT-PCR for the presence of lyssavirus RNA in triplicate and singly for host &#946; -actin mRNA. Positive and negative controls are included at extraction and real-time RT-PCR steps of the protocol. Data analysis involves manual adjustment of the thresholds to standardize Ct values across instrument runs. Positive results are determined by the presence of typical amplification in the pan-lyssavirus assay (Ct &#8804; 35). Negative results are determined by the absence of typical amplification in the pan-lyssavirus assay and detection of host &#946;-actin mRNA (Ct &#8804; 33). Observation of values outside of these ranges or failure of the assay controls can invalidate the run or result in inconclusive results for a specimen. The protocol should be closely followed to ensure high assay sensitivity and specificity. Procedural modifications can affect assay performance and lead to false positive, false negative, or uninterpretable results.

This protocol demonstrates the pan-lyssavirus LN34 real-time reverse transcription-polymerase chain reaction (RT-PCR) assay from tissue collection to result interpretation, including updates to primer sequences and formulations to improve assay performance for some non-rabies lyssaviruses and lagomorphs. We also demonstrate the assay setup for a single-well LN34 multiplexed (LN34M) format.

Rabies is a fatal zoonotic disease caused by Lyssavirus rabies (RABV) and related negative strand RNA viruses from the Lyssavirus genus (family ...

Biology

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

Source:&#160;<a style='text-decoration:none;' href='https://app.jove.com/v/59402/high-resolution-3d-imaging-of-rabies-virus-infection-in-solvent-cleared-brain-tissue'><span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>Zaeck, L.,&#160;et al.<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue.&#160;J. Vis. Exp.<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> (2019).</a> &#160;This video demonstrates the process of preparing and imaging mouse brain tissue infected with the rabies virus (RABV) to evaluate infection progression. It outlines key steps, including tissue staining with fluorescent markers, dehydration, clearing, and mounting for confocal laser scanning microscopy, culminating in the generation of a three-dimensional representation.

Source:&#160;Zaeck, L.,&#160;et al. High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue.&#160;J. Vis. Exp. ...

JoVE Encyclopedia of Experiments

Neuroimaging

Neurodegeneration & Disease Monitoring

Detection of Viral Proteins in Rabies Virus-Infected Mouse Brain Tissue

Collect fixed brain sections from intracerebral rabies virus-infected mice at defined postmortem intervals.&#160;
The virus infects pyramidal neurons in the cerebral cortex, and expressed viral proteins localize within intracytoplasmic granular structures known as inclusion bodies.
Treat the sections with ammonium chloride to neutralize fixation-induced reactive groups and minimize background staining.
Immerse the sections in hydrogen peroxide to inactivate endogenous peroxidase and reduce nonspecific signals.
Apply a blocking solution containing a detergent to permeabilize cellular membranes and mask nonspecific binding sites.
Incubate with primary antibodies specific to the viral proteins.
Wash and incubate with biotin-conjugated secondary antibodies that bind to the primary antibodies.
Wash again and introduce peroxidase enzyme-conjugated avidin, which binds to biotin, followed by a chromogenic substrate that the enzyme converts into a colored product.
Visualize the immunoreactive inclusion bodies containing viral proteins using a microscope.
With an increase in postmortem time, neuronal degradation and the disappearance of inclusion bodies become evident.

Neuropathology

Infectious Diseases

An Immunohistochemistry Staining for the Detection of Rabies Virus Antigens

Source: Niezgoda, M. et al., <a href="https://app.jove.com/v/60138">Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues.</a> J. Vis. Exp. (2021)
This video demonstrates immunohistochemistry staining for rabies virus antigen detection in infected mouse brain tissue. The process includes deparaffinization and alcohol treatment, followed by proteolytic treatment to enhance antigen accessibility. Immunohistochemistry staining reveals red precipitates, confirming the presence of rabies antigens in the tissue section.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Formalin fixation of tissues

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High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

Summary

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A Rat Model of EcoHIV Brain Infection

A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales

Postmortem Diagnosis of Rabies in Animals by the Updated, Multiplexed LN34 Real-Time Reverse Transcription-Polymerase Chain Reaction Assay

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

Detection of Viral Proteins in Rabies Virus-Infected Mouse Brain Tissue

An Immunohistochemistry Staining for the Detection of Rabies Virus Antigens