Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

Melina Thetiot; R&#233;mi Ronzano; Marie-St&#233;phane Aigrot; Catherine Lubetzki; Anne Desmazi&#232;res

doi:10.3791/59163

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

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Neuroscience

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JoVE Journal Neuroscience

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

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09:41 min

March 20, 2019

DOI: 10.3791/59163-v

Melina Thetiot¹, Rémi Ronzano¹, Marie-Stéphane Aigrot¹, Catherine Lubetzki¹, Anne Desmazières¹

¹Sorbonne Université, Inserm, CNRS, Institut du Cerveau et de la Moelle épinière, ICM-GH Pitié-Salpétrière

Overview

This study presents a method for preparing organotypic slice cultures from mouse cerebellum, focusing on myelin sheath staining through immunohistochemistry. The approach allows for the investigation of myelination and remyelination mechanisms in the central nervous system, while preserving tissue architecture.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Developmental Biology

Background

Myelination is essential for proper neuronal function and development.
Research on myelination can advance understanding of demyelinating diseases.
Organotypic slice cultures bridge in vitro and in vivo studies.

Purpose of Study

To develop a method for studying myelination and remyelination processes.
To provide a model that is accessible and effective for live imaging studies.
To enable quantitative approaches for drug screening experiments related to myelination.

Methods Used

Organotypic slice cultures were prepared from mouse cerebellum.
The model includes postnatal mice, with techniques for demyelination and myelination induction.
Immunohistochemistry was used to analyze myelin sheath formation.
Critical steps include tissue isolation, slicing, and fixation protocols.

Main Results

Cerebellar slices demonstrated spontaneous remyelination after LPC treatment.
Myelination of Purkinje cells was achieved mostly within one week in culture.
Results validate the model for studying myelin dynamics.

Conclusions

This method facilitates live imaging studies and provides a systematic approach to investigate myelination.
It enhances understanding of neuronal mechanisms and can apply to drug screening for demyelinating conditions.

Frequently Asked Questions

What are the advantages of organotypic slice cultures?

They preserve tissue architecture and allow for a combination of in vitro and in vivo methodologies.

How is the biological model implemented?

Organotypic slices are derived from mouse cerebellum, allowing for examination of myelination processes.

What types of data are obtained from this method?

Data include immunohistochemical analysis of myelination and observations on remyelination dynamics.

How can this method be adapted for drug screening?

The model allows for the introduction of pharmacological agents to assess their effects on myelination and remyelination.

What are the key limitations of this approach?

Considerations include the time required for culture preparation and the need for specific reagents for immunohistochemistry.

Here we present a method to prepare organotypic slice cultures from mouse cerebellum and myelin sheath staining by immunohistochemistry suitable for investigating mechanisms of myelination and remyelination in the central nervous system.

This method allows to study the basic mechanism of myelination and remyelination at the cellular and tissular level. This technique is at the crossroad between primary cultures and in vivo approaches. It is a fast and accessible model with the main advantage of preserving the tissue architecture.

Demonstrating the procedure will be Melina Thetiot, a postdoctoral researcher and Remi Ronzano, a PhD student from my laboratory. To begin, insert a small scissors gently into the foramen magnum and cut the skull by making one lateral incision towards the side, and then cut all around the head skull. To retrieve the dorsal part of the skull, use a fine, straight forceps to carefully lift the dorsal part of the skull.

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