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

Imaging 3D ad alta risoluzione dell'infezione da virus della rabbia nel tessuto cerebrale con la ...

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JoVE Journal Neuroscience

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

Imaging 3D ad alta risoluzione dell'infezione da virus della rabbia nel tessuto cerebrale con la rimozione del solvente

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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

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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.

Nuove tecniche di sgombero dei tessuti immunostaining come l'ultima imaging 3D di organi autorizzati dai solventi consentono la visualizzazione 3D dell'infezione cerebrale da virus del virus della rabbia e del suo complesso ambiente cellulare. Le fette spesse di tessuto cerebrale etichettate con anticorpi sono rese otticamente trasparenti per aumentare la profondità di imaging e consentire l'analisi 3D mediante microscopia a scansione laser confocale.

Questo protocollo consente la visualizzazione dell'infezione da virus e fornisce approfondimenti approfonditi sulla risoluzione spaziotemporale dell'ambiente di infezione e sul contesto cellulare circostante. L'applicabilità dell'immunosocienza relativa all'imaging dei tessuti profondi non solo consente il rilevamento di virus, ma di fatto consente anche la differenziazione di varie sottopopolazioni cellulari utilizzando i rispettivi marcatori cellulari. Per fissare il tessuto per l'immunostaining posizionare i campioni cerebrali in un rapporto uno-10 del 4% di paraformaldeide in PBS per campionare il volume del tessuto per almeno 48 ore a quattro gradi Celsius.

Al termine del periodo di fissazione lavare i campioni di tessuto tre volte in PBS per almeno 30 minuti per lavaggio prima di trasferire i campioni in azide di sodio al 02% in PBS a quattro gradi Celsius. Quindi utilizzare un vibratomo impostato su una velocità di avanzamento della lama da 0,3 a 0,5 millimetri al secondo per sessare i tessuti in sezioni spesse da un millimetro. Conservare le sezioni in azide fresco allo 02% di sodio in PBS a quattro gradi Celsius.

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