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

Luca Zaeck; Madlin Potratz; Conrad  M. Freuling; Thomas M&#252;ller; Stefan Finke

doi:10.3791/59402

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

JoVE Journal
Neuroscience

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JoVE Journal Neuroscience

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

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09:42 min

April 30, 2019

DOI: 10.3791/59402-v

Luca Zaeck¹, Madlin Potratz¹, Conrad M. Freuling¹, Thomas Müller¹, Stefan Finke¹

¹Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut,Federal Research Institute for Animal Health

Overview

This study explores novel immunostaining-compatible tissue clearing techniques that enable 3D visualization of rabies virus brain infection and its intricate cellular environment. The method enhances imaging depth, facilitating thorough analysis with confocal laser scanning microscopy.

Key Study Components

Area of Science

Neuroscience
Immunology
Tissue Imaging

Background

Understanding viral brain infections is crucial for neuroscience research.
Existing imaging techniques often face challenges with tissue opacity.
Immunostaining can differentiate various cellular subpopulations.
Recent advances in tissue clearing have improved imaging capabilities.

Purpose of Study

To visualize rabies virus infection in brain tissues.
To analyze the spatiotemporal resolution of the infection environment.
To enhance capabilities of deep tissue imaging through immunostaining.

Methods Used

The study utilizes immunostaining-compatible tissue clearing techniques.
Mouse brain tissue samples are employed as the biological model.
Immunostaining procedures allow visualization of virus phosphoprotein layers.
Various incubation and washing steps are conducted for optimal imaging.
The cleared tissues are captured using confocal laser scanning microscopy, enabling 3D analysis.

Main Results

The method successfully visualizes layers of infected neuronal cells.
High-resolution imaging correlates antigen abundance and distribution within cells.
Seamless 3D projections showcase the complexity of viral infections.
Detailed insights enhance understanding of infection processes at the cellular level.

Conclusions

This study demonstrates an effective technique for visualizing viral brain infections.
It underlines the importance of immunostaining in facilitating deep tissue imaging.
Findings contribute to a better understanding of neuronal mechanisms in response to infections.

Frequently Asked Questions

What are the advantages of the immunostaining-compatible clearing method?

This method allows for deep tissue imaging while preserving cellular structures, enabling detailed analysis of complex environments sparked by infections.

How is the rabies virus infection simulated in this study?

Infection is simulated through the introduction of the virus into mouse brain tissue, followed by extensive immunostaining procedures to reveal cellular interactions.

What types of data can be obtained through this method?

The method provides high-resolution images that reveal the abundance and distribution of viral antigens and cellular subpopulations within the brain tissue.

How can this method be applied in future research?

This approach can be adapted for studying other viral infections or diseases affecting the brain, allowing researchers to visualize interactions at a cellular level.

What are the limitations of the tissue clearing technique?

The technique may require optimization for different types of tissues or pathogens, and the extensive duration of the imaging process can limit throughput.

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.

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.

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