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.
Method Article
Three-Dimensional Visualization of a Rabies Virus Infection in Mouse Brain Tissue
May 29th, 2025
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In This Article
Abstract
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Protocol
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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
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Results
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Reagents | |||
| Benzyl alcohol | Alfa Aesar | 41218 | Clearing reagent |
| Benzyl benzoate | Sigma-Aldrich | BB6630-500ML | Clearing reagent |
| Dimethyl sulfoxide | Carl Roth | 4720.2 | Various buffers |
| Diphenyl ether | Sigma-Aldrich | 240834-100G | Clearing reagent |
| DL-α-Tocopherol | Alfa Aesar | A17039 | Antioxidant |
| Donkey serum | Bio-Rad | C06SBZ | Blocking reagent |
| Glycine | Carl Roth | 3908.2 | Background reduction |
| Goat serum | Merck | S26-100ML | Blocking reagent |
| Heparin sodium salt | Carl Roth | 7692.1 | Background reduction |
| Hydrogen peroxide solution (30 %) | Carl Roth | 8070.2 | Sample bleaching |
| Methanol | Carl Roth | 4627.4 | Sample pretreatment |
| Paraformaldehyde | Carl Roth | 335.3 | Crystalline powder to make fixative solution |
| Sodium azide | Carl Roth | K305.1 | Prevention of microbial growth in stock solutions |
| tert-Butanol | Alfa Aesar | 33278 | Sample dehydration for tissue clearing |
| TO-PRO-3 | Thermo Fisher | T3605 | Nucleic acid stain |
| Triton X-100 | Carl Roth | 3051.2 | Detergent |
| Tween 20 | AppliChem | A4974,0500 | Detergent |
| Miscellaneous | |||
| 5 mL reaction tubes | Eppendorf | 30119401 | Sample tubes |
| Coverslip, circular (diameter: 22 mm) | Marienfeld | 111620 | Part of imaging chamber |
| Coverslip, circular (diameter: 30 mm) | Marienfeld | 111700 | Part of imaging chamber |
| Hypodermic needle (27 G x ¾” [0.40 mm x 20 mm]) | B. Braun | 4657705 | Filling of the imaging chamber with clearing solution |
| RTV-1 silicone rubber | Wacker | Elastosil E43 | Adhesive for the assembly of the imaging chamber |
| Ultimaker CPE 2.85 mm transparent | Ultimaker | 8718836374869 | Copolyester filament for 3D printer to print parts of the imaging chamber |
| Technical equipment and software | |||
| 3D printer | Ultimaker | Ultimaker 2+ | Printing of imaging chamber |
| Automated water immersion system | Leica | 15640019 | Software-controlled water pump |
| Benchtop orbital shaker | Elmi | DOS-20M | Sample incubation at room temperature (~ 150 rpm) |
| Benchtop orbital shaker, heated | New Brunswick Scientific | G24 Environmental Shaker | Sample incubation at 37 °C (~ 150 rpm) |
| Confocal laser scanning microscope | Leica | DMI 6000 TCS SP5 | Inverted confocal microscope for sample imaging |
| Fiji | NIH (ImageJ) | open source software (v1.52h) | Image processing package based on ImageJ |
| Long working distance water immersion objective | Leica | 15506360 | HC PL APO 40x/1.10 W motCORR CS2 |
| Vibratome | Leica | VT1200S | Sample slicing |
| Workstation | Dell | Precision 7920 | CPU: Intel Xeon Gold 5118 |
| GPU: Nvidia Quadro P5000 | |||
| RAM: 128 GB 2666 MHz DDR4 | |||
| SSD: 2 TB | |||
| Primary antibodies | |||
| Goat anti-RABV N | Friedrich-Loeffler-Institut | Monospecific polyclonal goat anti-RABV N serum, generated by goat immunization with baculovirus-expressed and His-tag-purified RABV nucleoprotein N | |
| Dilution: 1:400 | |||
| Rabbit anti-GFAP | Dako | Z0334 | Polyclonal antibody (RRID:AB_10013382) |
| Dilution: 1:100 | |||
| Rabbit anti-MAP2 | Abcam | ab32454 | Polyclonal antibody (RRID:AB_776174) |
| Dilution: 1:250 | |||
| Rabbit anti-RABV P 160-5 | Friedrich-Loeffler-Institut | 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 | |||
| Secondary antibodies | |||
| Donkey anti-goat IgG | Thermo Fisher Scientific | depending on conjugated fluorophore | Highly cross-absorbed |
| Dilution: 1:500 | |||
| Donkey anti-mouse IgG | Thermo Fisher Scientific | depending on conjugated fluorophore | Highly cross-absorbed |
| Dilution: 1:500 | |||
| Donkey anti-rabbit IgG | Thermo Fisher Scientific | depending on conjugated fluorophore | Highly cross-absorbed |
| Dilution: 1:500 | |||
| Goat anti-mouse IgG | Thermo Fisher Scientific | depending on conjugated fluorophore | Highly cross-absorbed |
| Dilution: 1:500 | |||
| Goat anti-rabbit IgG | Thermo Fisher Scientific | depending on conjugated fluorophore | Highly cross-absorbed |
| Dilution: 1:500 | |||
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