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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical conduction. Myelin alteration has been shown to occur in various neurological diseases, where it is associated with functional deficits. Here, we provide a detailed description of an ex vivo model consisting of mouse organotypic cerebellar slices, which can be maintained in culture for several weeks and further be labeled to visualize myelin.

Introduction

Neurons are highly polarized cells, which comprise a somato-dendritic compartment that receives inputs from its environment and an axon that ensures the generation and propagation of electrical impulses to other cells. Rapid propagation and timely delivery of information is essential for the proper functioning of the nervous system. In vertebrates, it is facilitated by myelination, which allows increasing axonal conduction velocity1. Myelin is a specialized structure formed by compacted layers of plasma membrane generated by the myelinating glia, namely oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral ner....

Protocol

All work involving animals complied with institutional policies and guidelines established by the UPMC, INSERM and the French and European Community Council Directive 86/609/EEC.

1. Preparation of Culture Medium and Culture Inserts (Hands-on Time ≈ 10–15 min)

NOTE: Perform this step in a flow culture hood under sterile conditions

  1. Prepare 40 mL of culture medium, which consists of 50% BME, 25% Hank’s Balanced Salt Soluti.......

Representative Results

Examples of representative myelin immunostainings in organotypic cerebellar slices obtained from P9-P10 C57black6 wild-type (WT) (Figure 2A), as well as PLP-GFP transgenic mice (Figure 2B), together with Purkinje cells staining. Cerebellar slices myelinate from the white matter tracks region of the slices towards the periphery of the folia and myelination of the Purkinje cells is mostly achieved after 6 to 7 DIV. At 7 DIV, the in.......

Discussion

Here, we detail a protocol to generate an ex vivo model corresponding to the mouse cerebellar organotypic slice cultures, adapted from previously published methods15,16,19 and the subsequent myelin immunostaining of these preparations. This strategy offers the possibility to visualize myelin components with a high-resolution in both healthy and pathological states.

Cerebellar organotypic slice culture.......

Acknowledgements

We thank Dr. Sean Freeman, Dr. Nathalie Sol-Foulon and Dr. Thomas Roux for valuable comments on the manuscript. This work was funded by INSERM, ICM, ARSEP Grants R13123DD, ANR R17127DD (to A.D.) and FRM fellowship, SPF20110421435 (to A.D.), FDT20170437332 (to M.T.). We thank the CELIS cell culture facility and the icm.Quant imaging platform.

....

Materials

NameCompanyCatalog NumberComments
BME mediumThermoFisher Scientific41010026
Hank’s Balanced Salt Solution (10X HBSS)ThermoFisher Scientific14180046
GlutaMAX (100X)ThermoFisher Scientific35050038
Heat-inactivated Horse SerumThermoFisher Scientific26050088
Penicillin–Streptomycin (10.000 IU/mL)ThermoFisher Scientific15140122
Gey’s Balanced Salt SolutionSigma AldrichG9779-500ML
D-Glucose Solution (45%)Sigma AldrichG8769-100ML
Lysophosphatidylcholine (LPC)Sigma AldrichL4129-100MG
Paraformaldehyde (PFA)Electron Microscopy Sciences15714
Absolute ethanol (100% ethanol)VWR Chemicals20821.330Cooled at -20°C
Triton® X-100Sigma AldrichX100-500ML
10% Normal Goat Serum (NGS)ThermoFisher Scientific500622
Phosphate Buffer SolutionEuroMEDEXET330-ApH 7.4
Anti-GFP Antibody (Polyclonal, Chicken)Merck Millipore06-896Dilution 1/300
Anti-Myelin Basic Protein (MBP) Antibody (Polyclonal, Chicken)Merck MilliporeAB9348Dilution 1/150
Anti-Myelin Basic Protein (MBP) Antibody (Monoclonal, Mouse IgG2b)Merck MilliporeNE1019Dilution 1/200
Anti-PLP Antibody (Rat, Hybridoma)Gift from Dr. K. Ikenaka; Okasaki, JapanDilution 1/5 to 1/10
Anti-Sodium Channel, Pan Antibody (Monoclonal, Mouse IgG1, clone K58/35)Sigma AldrichS8809Dilution 1/150
Anti-Caspr Antibody (Polyclonal, Rabbit)Abcamab34151Dilution 1/500
Goat Secondary Antibodies conjugated to Alexa Fluor 488, 594, 647 or 405ThermoFisher ScientificDilution 1/500
FluoromountSouthernBiotech0100-20
Tissue chopperMcIlwain
Razor blades
Large scissorsF.S.T14101-14
Small scissorsF.S.T91500-09
Fine-straight forcepsF.S.T91150-20
Curved-fine forcepsF.S.T11297-00
Cell culture dishes (60-mm and 100-mm)TPP
4-, 6-well culture platesTPP
Millicell culture inserts (0,4 µm, 30-mm diameter)Merck MilliporePICM0RG50
Cell culture incubator37°C, 5% CO2
Fine-end pipette tipsDutscher134000CL
Wide-bore pipette tipsThermoFisher Scientific2079G
Sterile syringeTerumo Europe20 or 50 mL
Sterile syringe filtersTerumo Europe0.22 µm
ScalpelSwann-Morton0510
Brush
Microscope slidesRS France76 x 26 x 1.1 mm
Glass coverslipsRS France22 x 22 mm
Kimtech Sciences Tissue WipersKimberly-Clark Professional5511
Binocular microscope

References

  1. Monje, M. Myelin Plasticity and Nervous System Function. Annual Review Neuroscience. 41, 61-76 (2018).
  2. Desmazières, A., Sol-Foulon, N., Lubetzki, C. Changes at the nodal and perinodal axonal domains: a basis fo....

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