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.).&#160;We thank the CELIS cell culture facility and the icm.Quant imaging platform.

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 &#8776; 10&#8211;15 min)
NOTE: Perform this step in a flow culture hood under sterile conditions
<ol>
	<li>Prepare 40 mL of culture medium, which consists of 50% BME, 25% Hank&#8217;s Balanced Salt Soluti...

<ol>
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G., Stelzer, E. H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+third+dimension+bridges+the+gap+between+cell+culture+and+live+tissue.">The third dimension bridges the gap between cell culture and live tissue.</a> Nature Reviews Molecular Cell Biology. 8, 839-845 (2007).</li><li>Medina-Rodr&#237;guez, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promoting+in+vivo+remyelination+with+small+molecules:+a+neuroreparative+pharmacological+treatment+for+Multiple+Sclerosis.">Promoting in vivo remyelination with small molecules: a neuroreparative pharmacological treatment for Multiple Sclerosis.</a> Science Reports. 7, (2017).</li><li>Spassky, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+early+steps+of+oligodendrogenesis:+insights+from+the+study+of+the+plp+lineage+in+the+brain+of+chicks+and+rodents.">The early steps of oligodendrogenesis: insights from the study of the plp lineage in the brain of chicks and rodents.</a> Developmental Neurosciences. 23, 318-326 (2001).</li><li>Yuan, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+green+fluorescent+protein+in+the+oligodendrocyte+lineage:+a+transgenic+mouse+for+developmental+and+physiological+studies.">Expression of the green fluorescent protein in the oligodendrocyte lineage: a transgenic mouse for developmental and physiological studies.</a> Journal of Neuroscience Research. 70, 529-545 (2002).</li><li>Gibson, E. 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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...

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 nervous system (PNS). Both in the CNS and the PNS, axoglial interactions drive the formation of specialized axonal domains: the nodes of Ranvier and their surrounding domains, the paranodes, and juxtaparanodes2. The axonal segments insulated by myelin, or internodes, alternate with the nodes of Ranvier, which correspond to small unmyelinated domains enriched in voltage-gated sodium channels (Nav). The high concentration and rapid activation of Nav channels at the nodes of Ranvier allow the regeneration of action potentials, and together with the insulating properties of the myelin sheaths, ensure the efficient and fast saltatory conduction of the nerve impulse along the axon3.
In addition to its role in accelerating the conduction velocity of the nerve impulse, myelinating glia provides metabolic support to the axon, preserving its long-term integrity and participating in its survival4,5. Furthermore, it has become clear in recent years that myelin is dynamically modulated throughout life, thus presumably participating in the regulation and plasticity of various nervous system functions. Adjustments of the distribution, number, length, and thickness of myelin sheaths along axons might thus represent a novel way to finely tune various networks6,7,8. Therefore, the evolutionary acquisition of myelin is a key process for sensory, motor and cognitive functions and the perturbation of the interaction between axons and glia is increasingly considered as contributing to the developmental or acquired neurological diseases9.
Myelin composition has been characterized, with the specific feature of a high proportion of lipids (70%) compared to proteins (30%) in contrast to other cellular membranes10. However, unlike myelin lipids, most of myelin proteins are specific to myelin, including myelin basic protein (MBP), proteolipid protein (PLP), 2&#39;,3&#39;-cyclic nucleotide 3&#39;-phosphodiesterase (CNP), myelin-associated glycoprotein (MAG), myelin-oligodendrocyte glycoprotein (MOG), PMP-22 and P010. Various histological methods to stain myelin exist based on its lipid composition, such as Luxol fast blue11, Sudan Black B12, Baker&#39;s acid hematin method13, as well as silver staining14. Nevertheless, these approaches do not always allow for an adequate contrast and resolution to visualize individual fibers. An alternative approach to detect myelin is through immunohistochemistry directed against myelin proteins. Various antibodies target myelin-specific antigens with a high specificity and can be used routinely to detect myelinated structures. The antibody-antigen interaction can be further revealed using a secondary antibody coupled to a fluorophore directed against the primary antibody and visualized with adequate fluorescence microscopy. Here, we describe an immunochemical protocol to stain myelin on ex vivo cerebellar slices, a model which allows for a good preservation of the nervous tissue architecture. In addition, the organization and size of the Purkinje cells (the sole myelinated neuron of the cerebellum) make them a classical model for electrophysiological studies and they are similarly ideal to perform fixed or live-imaging studies.
The cerebellar slices are generated from P9-P10 mice, a time corresponding to the early onset of Purkinje cell myelination, a process that is mostly achieved by one week ex vivo (6-7 days in vitro, DIV)15. Furthermore, this model is adapted to investigate demyelinating disorders such as multiple sclerosis (MS), as an extensive demyelination can be induced in cerebellar slices using the myelinotoxic compound lysophosphatidylcholine (or lysolecithin, LPC), which is followed by a spontaneous remyelination16,17. Endogenous remyelination takes place from two days after LPC removal from the culture medium and is almost complete a week post treatment.
The completion of this protocol takes approximatively 3 weeks, including half a day for cerebellar slice cultures preparation, a week to obtain fully myelinated slices, followed by 2 days to reach the peak of demyelination and another week for their full remyelination. In addition, immunohistochemistry can be completed in 2 days. The protocol described here is adapted to a standard litter of 6 mice pups and needs to be adapted regarding the number of animals used for the planned experiment.

<table>
	<tbody>
		<tr>
			<td>BME medium</td>
			<td>ThermoFisher Scientific</td>
			<td>41010026</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution (10X HBSS)</td>
			<td>ThermoFisher Scientific</td>
			<td>14180046</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX (100X)</td>
			<td>ThermoFisher Scientific</td>
			<td>35050038</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat-inactivated Horse Serum</td>
			<td>ThermoFisher Scientific</td>
			<td>26050088</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin&#8211;Streptomycin (10.000 IU/mL)</td>
			<td>ThermoFisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Gey&#8217;s Balanced Salt Solution</td>
			<td>Sigma Aldrich</td>
			<td>G9779-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose Solution (45%)</td>
			<td>Sigma Aldrich</td>
			<td>G8769-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysophosphatidylcholine (LPC)</td>
			<td>Sigma Aldrich</td>
			<td>L4129-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Electron Microscopy Sciences</td>
			<td>15714</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute ethanol (100% ethanol)</td>
			<td>VWR Chemicals</td>
			<td>20821.330</td>
			<td>Cooled at -20&#176;C</td>
		</tr>
		<tr>
			<td>Triton&#174; X-100</td>
			<td>Sigma Aldrich</td>
			<td>X100-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Normal Goat Serum (NGS)</td>
			<td>ThermoFisher Scientific</td>
			<td>500622</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Solution</td>
			<td>EuroMEDEX</td>
			<td>ET330-A</td>
			<td>pH 7.4</td>
		</tr>
		<tr>
			<td>Anti-GFP Antibody (Polyclonal, Chicken)</td>
			<td>Merck Millipore</td>
			<td>06-896</td>
			<td>Dilution 1/300</td>
		</tr>
		<tr>
			<td>Anti-Myelin Basic Protein (MBP) Antibody (Polyclonal, Chicken)</td>
			<td>Merck Millipore</td>
			<td>AB9348</td>
			<td>Dilution 1/150</td>
		</tr>
		<tr>
			<td>Anti-Myelin Basic Protein (MBP) Antibody (Monoclonal, Mouse IgG2b)</td>
			<td>Merck Millipore</td>
			<td>NE1019</td>
			<td>Dilution 1/200</td>
		</tr>
		<tr>
			<td>Anti-PLP Antibody (Rat, Hybridoma)</td>
			<td>Gift from Dr. K. Ikenaka; Okasaki, Japan</td>
			<td></td>
			<td>Dilution 1/5 to 1/10</td>
		</tr>
		<tr>
			<td>Anti-Sodium Channel, Pan Antibody (Monoclonal, Mouse IgG1, clone K58/35)</td>
			<td>Sigma Aldrich</td>
			<td>S8809</td>
			<td>Dilution 1/150</td>
		</tr>
		<tr>
			<td>Anti-Caspr Antibody (Polyclonal, Rabbit)</td>
			<td>Abcam</td>
			<td>ab34151</td>
			<td>Dilution 1/500</td>
		</tr>
		<tr>
			<td>Goat Secondary Antibodies conjugated to Alexa Fluor 488, 594, 647 or 405</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td>Dilution 1/500</td>
		</tr>
		<tr>
			<td>Fluoromount</td>
			<td>SouthernBiotech</td>
			<td>0100-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue chopper</td>
			<td>McIlwain</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Large scissors</td>
			<td>F.S.T</td>
			<td>14101-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Small scissors</td>
			<td>F.S.T</td>
			<td>91500-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine-straight forceps</td>
			<td>F.S.T</td>
			<td>91150-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved-fine forceps</td>
			<td>F.S.T</td>
			<td>11297-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dishes (60-mm and 100-mm)</td>
			<td>TPP</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4-, 6-well culture plates</td>
			<td>TPP</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell culture inserts (0,4 &#181;m, 30-mm diameter)</td>
			<td>Merck Millipore</td>
			<td>PICM0RG50</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td></td>
			<td></td>
			<td>37&#176;C, 5% CO2</td>
		</tr>
		<tr>
			<td>Fine-end pipette tips</td>
			<td>Dutscher</td>
			<td>134000CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide-bore pipette tips</td>
			<td>ThermoFisher Scientific</td>
			<td>2079G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile syringe</td>
			<td>Terumo Europe</td>
			<td></td>
			<td>20 or 50 mL</td>
		</tr>
		<tr>
			<td>Sterile syringe filters</td>
			<td>Terumo Europe</td>
			<td></td>
			<td>0.22 &#181;m</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Swann-Morton</td>
			<td>0510</td>
			<td></td>
		</tr>
		<tr>
			<td>Brush</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>RS France</td>
			<td></td>
			<td>76 x 26 x 1.1 mm</td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td>RS France</td>
			<td></td>
			<td>22 x 22 mm</td>
		</tr>
		<tr>
			<td>Kimtech Sciences Tissue Wipers</td>
			<td>Kimberly-Clark Professional</td>
			<td>5511</td>
			<td></td>
		</tr>
		<tr>
			<td>Binocular microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation and immunostaining of myelinating organotypic cerebellar slice cultures

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.

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...

Watch this Scientific Journal Video about Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures at JoVE.com

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

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 ...

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.Then carefully introduce the forceps between the ventral skull and the brain. Gently flip out the brain and cut the optic and trigeminal nerves with small scissors. Next, turn the head or the dorsal part of the skull upside down just above a 60 millimeter cell culture dish containing ice cold dissection medium to help the brain to drop by gravity.Using fine forceps, orientate the brain with the dorsal side facing up and the ventral side lying down. Under the binocular microscope, use the fine, straight forceps to immobilize the brain on the forebrain side. Then, separate the hind brain from the rest of the brain.Cut the cerebellar peduncles underneath the cerebellum to separate the cerebellum from the rest of the hind brain. Once the cerebellum is isolated, use fine, straight forceps to carefully tear away the meninges. Hold the cerebellum gently with the fine, curved forceps and place it with the dorsal side up onto the plastic platform, perpendicularly to the chopper razor blade.Next, with a sterile, thin end pipette tip attached to a one milliliter pipette, aspirate any access of dissection medium around the cerebellum. Then, with a tissue chopper, slice 300 micrometer thick saggital sections of the cerebellum. Next, add a drop of dissection medium onto the sliced cerebellum gently.Then, with a wide boar pipette tip attached to the one milliliter pipette, slowly aspirate the sliced cerebellum and transfer it back into the 60 millimeter cell culture dish containing ice cold dissection medium. Next, use two fine, straight forceps to separate individual slices. Then, with a wide boar pipette tip attached to the one milliliter pipette, transfer up to four selected slices from the vermis, along with some dissection medium onto one culture insert of a six well plate.Remove any access of dissection medium around the slices using a thin end pipette tip. For demyelination, remove all culture medium below the culture inserts after six days in vitro, and replace it with one milliliter per well of the pre-warmed, fresh culture medium containing 0.5 milligram per milliliter LPC. Incubate for 15 to 17 hours at 37 degrees Celsius in 5%carbon dioxide environment.After incubation, wash the inserts by placing them in a 25 milliliter Petri dish containing one milliliter of pre-warmed culture medium. Then, immediately transfer the culture inserts in a new, six well plate, containing fresh, pre-warmed culture medium. To begin immunohistochemistry, first use forceps to lift culture inserts and remove the culture medium beneath them.Then, add two milliliters of 4%PFA in 1x PBS, ph 7.4, onto the membrane inserts to fix the cerebellar slices. After 30 minutes, wash the slices three times with two milliliters of 1x PBS for 10 minutes each wash. Next, under the binocular microscope, using a 25x to 30x magnification, use a scalpel or a brush to gently detach the slices from the membrane inserts.Then, use a brush to transfer the floating slices into the wells of a four well plate, containing 1x PBS to limit antibody consumption. Then, aspirate the PBS from each well and incubate the slices in pre-cooled 100%ethanol at minus 20 degrees Celsius for 15 to 20 minutes. After incubation, aspirate the 100%ethanol and wash the slices briefly with 1x PBS.Then, wash them two times at room temperature with 1x PBS for 10 minutes each wash. To block non-specific antibody fixation sites, aspirate the PBS and incubate the slices in a solution containing 1x PBS, 5%NGS, and 0.3%non ionic detergent at room temperature for 30 to 45 minutes. Next, add primary antibodies diluted in the blocking solution and incubate the slices at four degrees Celsius overnight.After overnight incubation, wash the slices three times with 1x PBS for 10 minutes each wash. Then add the secondary antibodies, diluted in the blocking solution, at one to 500 dilution ratio and incubate the slices at room temperature in the dark for three hours. After incubation with the secondary antibody, wash the slices three times in 1x PBS in the dark for 10 minutes each wash.Next, under a binocular microscope, place 100 microleters of 1x PBS on the slide and with a brush, transfer the slices into the PBS and flatten them on the slide. Remove any access of PBS. Finally, place a drop of mounting medium directly onto the glass cover slip and gently cover the slices.Myelin immunostainings in organotypic cerebellar slices, obtained from post natal days 9 to 10 C57 black six wild type mice were observed at 11 days in vitro with nodes of ronviae enriched in voltage gated sodium channels, flanked by the paranodal axoglial junction domain. Same results were observed for PLP-GFP transgenic mice. Myelination of the parkengy cells is mostly achieved after one week in culture.LPC treatment fully demyelinates the slices, which remyelinate spontaneously and are fully myelinated six days post demyelination. This procedure should take 25 minute maximum. It is really the most critical point.Organotypic slice culture are suitable for live imaging study of myelination dynamics. It can also be used for targeting drug screening experiments. This technique allows an easy, quantitative approach to study myelination and remyelination processes.Experimenters are to follow the basic safety procedures applying to animal welfare, excessive and sharp myelination.

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Research

JoVE Journal

Neuroscience

10.7K vistas.  Sorbonne Université, Inserm, CNRS, Institut du Cerveau et de la Moelle épinière, ICM-GH Pitié-Salpétrière. Este método permite estudiar el mecanismo básico de mielinización y reeyelinación a nivel celular y tisular. Esta técnica está en la encrucijada entre las culturas primarias y los enfoques in vivo. Es un modelo rápido y accesible con la principal ventaja de preservar la arquitectura tisular.Demostrando el procedimiento estarán Melina Thetiot, investigadora postdoctoral y Remi Ronzano, estudiante de doctorado de mi laboratorio. Para comenzar, inserte unas tijeras pequeñas suave...