Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

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.......

Discussion

Here, we detail a protocol to generate an ex vivo model corresponding to the mouse cerebellar organotypic slice cultures, adapted from previously published methods^15,16,19 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.......

Materials

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