Name: high-resolution 3d imaging of rabies virus infection in solvent-cleared brain tissue
Uploaded: 2019-04-30T13:00:00
Description: Watch this Scientific Journal Video about High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue at JoVE.com

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: Thi...

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The resurgence and further development of tissue clearing techniques in recent years2,3,4,5,6,7,8,9,10,11,12,13,...

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 border="0" cellpadding="0" cellspacing="0" style="width:827px;" width="827">
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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Reagents</td>
			<td style="width:119px;"></td>
			<td style="width:160px;"></td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Benzyl alcohol</td>
			<td style="width:119px;">Alfa Aesar</td>
			<td style="width:160px;">41218</td>
			<td style="width:297px;">Clearing reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Benzyl benzoate</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:160px;">BB6630-500ML</td>
			<td style="width:297px;">Clearing reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Dimethyl sulfoxide</td>
			<td style="width:119px;">Carl Roth</td>
			<td style="width:160px;">4720.2</td>
			<td style="width:297px;">Various buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Diphenyl ether</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:160px;">240834-100G</td>
			<td style="width:297px;">Clearing reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">DL-&#945;-Tocopherol</td>
			<td style="width:119px;">Alfa Aesar</td>
			<td style="width:160px;">A17039</td>
			<td style="width:297px;">Antioxidant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Donkey serum</td>
			<td style="width:119px;">Bio-Rad</td>
			<td style="width:160px;">C06SBZ</td>
			<td style="width:297px;">Blocking reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Glycine</td>
			<td style="width:119px;">Carl Roth</td>
			<td style="width:160px;">3908.2</td>
			<td style="width:297px;">Background reduction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Goat serum</td>
			<td style="width:119px;">Merck</td>
			<td style="width:160px;">S26-100ML</td>
			<td style="width:297px;">Blocking reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Heparin sodium salt</td>
			<td style="width:119px;">Carl Roth</td>
			<td style="width:160px;">7692.1</td>
			<td style="width:297px;">Background reduction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Hydrogen peroxide solution (30 %)</td>
			<td style="width:119px;">Carl Roth</td>
			<td style="width:160px;">8070.2</td>
			<td style="width:297px;">Sample bleaching</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Methanol</td>
			<td style="width:119px;">Carl Roth</td>
			<td style="width:160px;">4627.4</td>
			<td style="width:297px;">Sample pretreatment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Paraformaldehyde</td>
			<td style="width:119px;">Carl Roth</td>
			<td style="width:160px;">0335.3</td>
			<td style="width:297px;">Crystalline powder to make fixative solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Sodium azide</td>
			<td style="width:119px;">Carl Roth</td>
			<td style="width:160px;">K305.1</td>
			<td style="width:297px;">Prevention of microbial growth in stock solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">tert-Butanol</td>
			<td style="width:119px;">Alfa Aesar</td>
			<td style="width:160px;">33278</td>
			<td style="width:297px;">Sample dehydration for tissue clearing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">TO-PRO-3</td>
			<td style="width:119px;">Thermo Fisher</td>
			<td style="width:160px;">T3605</td>
			<td style="width:297px;">Nucleic acid stain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Triton X-100</td>
			<td style="width:119px;">Carl Roth</td>
			<td style="width:160px;">3051.2</td>
			<td style="width:297px;">Detergent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Tween 20</td>
			<td style="width:119px;">AppliChem</td>
			<td style="width:160px;">A4974,0500</td>
			<td style="width:297px;">Detergent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Miscellaneous</td>
			<td style="width:119px;"></td>
			<td style="width:160px;"></td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">5 mL reaction tubes</td>
			<td style="width:119px;">Eppendorf</td>
			<td style="width:160px;">0030119401</td>
			<td style="width:297px;">Sample tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Coverslip, circular (diameter: 22 mm)</td>
			<td style="width:119px;">Marienfeld</td>
			<td style="width:160px;">0111620</td>
			<td style="width:297px;">Part of imaging chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Coverslip, circular (diameter: 30 mm)</td>
			<td style="width:119px;">Marienfeld</td>
			<td style="width:160px;">0111700</td>
			<td style="width:297px;">Part of imaging chamber</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Hypodermic needle (27 G x &#190;&#8221; [0.40 mm x 20 mm])</td>
			<td style="width:119px;">B. Braun</td>
			<td style="width:160px;">4657705</td>
			<td style="width:297px;">Filling of the imaging chamber with clearing solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">RTV-1 silicone rubber</td>
			<td style="width:119px;">Wacker</td>
			<td style="width:160px;">Elastosil E43</td>
			<td style="width:297px;">Adhesive for the assembly of the imaging chamber</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Ultimaker CPE 2.85 mm transparent</td>
			<td style="width:119px;">Ultimaker</td>
			<td style="width:160px;">8718836374869</td>
			<td style="width:297px;">Copolyester filament for 3D printer to print parts of the imaging chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Technical equipment and software</td>
			<td style="width:119px;"></td>
			<td style="width:160px;"></td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">3D printer</td>
			<td style="width:119px;">Ultimaker</td>
			<td style="width:160px;">Ultimaker 2+</td>
			<td style="width:297px;">Printing of imaging chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Automated water immersion system</td>
			<td style="width:119px;">Leica</td>
			<td style="width:160px;">15640019</td>
			<td style="width:297px;">Software-controlled water pump</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Benchtop orbital shaker</td>
			<td style="width:119px;">Elmi</td>
			<td style="width:160px;">DOS-20M</td>
			<td style="width:297px;">Sample incubation at room temperature (~ 150 rpm)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Benchtop orbital shaker, heated</td>
			<td style="width:119px;">New Brunswick Scientific</td>
			<td style="width:160px;">G24 Environmental Shaker</td>
			<td style="width:297px;">Sample incubation at 37 &#176;C (~ 150 rpm)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Confocal laser scanning microscope</td>
			<td style="width:119px;">Leica</td>
			<td style="width:160px;">DMI 6000 TCS SP5</td>
			<td style="width:297px;">Inverted confocal microscope for sample imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Fiji</td>
			<td style="width:119px;">NIH (ImageJ)</td>
			<td style="width:160px;">open source software (v1.52h)</td>
			<td style="width:297px;">Image processing package based on ImageJ</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Long working distance water immersion objective</td>
			<td style="width:119px;">Leica</td>
			<td style="width:160px;">15506360</td>
			<td style="width:297px;">HC PL APO 40x/1.10 W motCORR CS2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Vibratome</td>
			<td style="width:119px;">Leica</td>
			<td style="width:160px;">VT1200S</td>
			<td style="width:297px;">Sample slicing</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:251px;">Workstation</td>
			<td style="width:119px;">Dell</td>
			<td style="width:160px;">Precision 7920</td>
			<td style="width:297px;">CPU: Intel Xeon Gold 5118 
			GPU: Nvidia Quadro P5000 
			RAM: 128 GB 2666 MHz DDR4 
			SSD: 2 TB</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Primary antibodies</td>
			<td style="width:119px;"></td>
			<td style="width:160px;"></td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:251px;">Goat anti-RABV N</td>
			<td style="width:119px;">Friedrich-Loeffler-Institut</td>
			<td style="width:160px;"></td>
			<td style="width:297px;">Monospecific polyclonal goat anti-RABV N serum, generated by goat immunization with baculovirus-expressed and His-tag-purified RABV nucleoprotein N 
			Dilution: 1:400</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Rabbit anti-GFAP</td>
			<td style="width:119px;">Dako</td>
			<td style="width:160px;">Z0334</td>
			<td style="width:297px;">Polyclonal antibody (RRID:AB_10013382) 
			Dilution: 1:100</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Rabbit anti-MAP2</td>
			<td style="width:119px;">Abcam</td>
			<td style="width:160px;">ab32454</td>
			<td style="width:297px;">Polyclonal antibody (RRID:AB_776174) 
			Dilution: 1:250</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:251px;">Rabbit anti-RABV P 160-5</td>
			<td style="width:119px;">Friedrich-Loeffler-Institut</td>
			<td style="width:160px;"></td>
			<td style="width:297px;">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) 
			Dilution: 1:1,000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Secondary antibodies</td>
			<td style="width:119px;"></td>
			<td style="width:160px;"></td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:251px;">Donkey anti-goat IgG</td>
			<td style="width:119px;">Thermo Fisher Scientific</td>
			<td style="width:160px;">depending on conjugated fluorophore</td>
			<td style="width:297px;">Highly cross-absorbed 
			Dilution: 1:500</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:251px;">Donkey anti-mouse IgG</td>
			<td style="width:119px;">Thermo Fisher Scientific</td>
			<td style="width:160px;">depending on conjugated fluorophore</td>
			<td style="width:297px;">Highly cross-absorbed 
			Dilution: 1:500</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:251px;">Donkey anti-rabbit IgG</td>
			<td style="width:119px;">Thermo Fisher Scientific</td>
			<td style="width:160px;">depending on conjugated fluorophore</td>
			<td style="width:297px;">Highly cross-absorbed 
			Dilution: 1:500</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:251px;">Goat anti-mouse IgG</td>
			<td style="width:119px;">Thermo Fisher Scientific</td>
			<td style="width:160px;">depending on conjugated fluorophore</td>
			<td style="width:297px;">Highly cross-absorbed 
			Dilution: 1:500</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:251px;">Goat anti-rabbit IgG</td>
			<td style="width:119px;">Thermo Fisher Scientific</td>
			<td style="width:160px;">depending on conjugated fluorophore</td>
			<td style="width:297px;">Highly cross-absorbed 
			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 ...

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 ...

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.

Research

JoVE Journal

Neuroscience

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

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 ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. ...