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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.
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.
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....
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
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.......
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.......
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.
....Name | Company | Catalog Number | Comments |
BME medium | ThermoFisher Scientific | 41010026 | |
Hank’s Balanced Salt Solution (10X HBSS) | ThermoFisher Scientific | 14180046 | |
GlutaMAX (100X) | ThermoFisher Scientific | 35050038 | |
Heat-inactivated Horse Serum | ThermoFisher Scientific | 26050088 | |
Penicillin–Streptomycin (10.000 IU/mL) | ThermoFisher Scientific | 15140122 | |
Gey’s Balanced Salt Solution | Sigma Aldrich | G9779-500ML | |
D-Glucose Solution (45%) | Sigma Aldrich | G8769-100ML | |
Lysophosphatidylcholine (LPC) | Sigma Aldrich | L4129-100MG | |
Paraformaldehyde (PFA) | Electron Microscopy Sciences | 15714 | |
Absolute ethanol (100% ethanol) | VWR Chemicals | 20821.330 | Cooled at -20°C |
Triton® X-100 | Sigma Aldrich | X100-500ML | |
10% Normal Goat Serum (NGS) | ThermoFisher Scientific | 500622 | |
Phosphate Buffer Solution | EuroMEDEX | ET330-A | pH 7.4 |
Anti-GFP Antibody (Polyclonal, Chicken) | Merck Millipore | 06-896 | Dilution 1/300 |
Anti-Myelin Basic Protein (MBP) Antibody (Polyclonal, Chicken) | Merck Millipore | AB9348 | Dilution 1/150 |
Anti-Myelin Basic Protein (MBP) Antibody (Monoclonal, Mouse IgG2b) | Merck Millipore | NE1019 | Dilution 1/200 |
Anti-PLP Antibody (Rat, Hybridoma) | Gift from Dr. K. Ikenaka; Okasaki, Japan | Dilution 1/5 to 1/10 | |
Anti-Sodium Channel, Pan Antibody (Monoclonal, Mouse IgG1, clone K58/35) | Sigma Aldrich | S8809 | Dilution 1/150 |
Anti-Caspr Antibody (Polyclonal, Rabbit) | Abcam | ab34151 | Dilution 1/500 |
Goat Secondary Antibodies conjugated to Alexa Fluor 488, 594, 647 or 405 | ThermoFisher Scientific | Dilution 1/500 | |
Fluoromount | SouthernBiotech | 0100-20 | |
Tissue chopper | McIlwain | ||
Razor blades | |||
Large scissors | F.S.T | 14101-14 | |
Small scissors | F.S.T | 91500-09 | |
Fine-straight forceps | F.S.T | 91150-20 | |
Curved-fine forceps | F.S.T | 11297-00 | |
Cell culture dishes (60-mm and 100-mm) | TPP | ||
4-, 6-well culture plates | TPP | ||
Millicell culture inserts (0,4 µm, 30-mm diameter) | Merck Millipore | PICM0RG50 | |
Cell culture incubator | 37°C, 5% CO2 | ||
Fine-end pipette tips | Dutscher | 134000CL | |
Wide-bore pipette tips | ThermoFisher Scientific | 2079G | |
Sterile syringe | Terumo Europe | 20 or 50 mL | |
Sterile syringe filters | Terumo Europe | 0.22 µm | |
Scalpel | Swann-Morton | 0510 | |
Brush | |||
Microscope slides | RS France | 76 x 26 x 1.1 mm | |
Glass coverslips | RS France | 22 x 22 mm | |
Kimtech Sciences Tissue Wipers | Kimberly-Clark Professional | 5511 | |
Binocular microscope |
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