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 Solution (1x), 25% heat-inactivated horse serum, supplemented with 2 mM glutamine derivative, 100 IU/mL penicillin-streptomycin and 4.5 mg/mL D-Glucose. Adjust the pH to 7.4.</li>
	<li>Filter-sterilize the medium through a 0.22 &#956;m filter adapted on a 50 mL plastic syringe. 
	NOTE: Culture medium can be stored at 4 &#176;C for at least a week.</li>
	<li>For a standard litter of 6 pups, use two sterile 6-well plates. For each plate, remove the cover and add 1 mL of culture medium per well.</li>
	<li>Place one culture insert into each individual well by holding the plastic edge with sterile forceps. Put the cover back on the plate. 
	NOTE: The cerebellar slices collected from one pup (6-8) can be dispatched on two membranes (3-4 cerebellar slices per membrane insert).</li>
</ol>
2. Preparation of Dissection Medium (Hands-on Time &#8776; 5 min)
NOTE: Perform this step in a flow culture hood under sterile conditions
<ol>
	<li>Prepare 100 mL of dissection medium, which consists of Gey&#8217;s Balanced Salt Solution supplemented with 4.5 mg/mL D-Glucose and 1x penicillin&#8211;streptomycin (100 IU/mL each).</li>
	<li>Filter-sterilize the medium through a 0.22 &#956;m filter unit. 
	NOTE: The dissection medium can be stored at 4 &#176;C for at least a month.</li>
	<li>Add 5 mL of cold dissection medium into 60 mm cell culture dishes. Prepare one 60 mm cell culture dish for each pup to be dissected.</li>
	<li>Keep the dissection dishes on ice until use.</li>
</ol>
3. Preparation of dissection material (Hands-on Time &#8776; 15 min)
<ol>
	<li>Clean the bench with 100% ethanol.</li>
	<li>Sterilize all dissection tools, a razor blade and the tissue chopper&#8217;s plastic platform with 100% ethanol.</li>
	<li>Fix the razor blade and the plastic platform on the tissue chopper and adjust the cutting thickness to 300 &#181;m.</li>
	<li>Make sure all the ethanol has evaporated before starting the dissection.</li>
</ol>
4. CerebellumDissection and Sections Preparation (Hands-on Time &#8776; 15&#8211;20 min per animal)
<ol>
	<li>Place one of the 60 mm cell culture dishes containing the ice-cold dissection medium under the binocular microscope and remove the top plate.</li>
	<li>Swab the head fur with 70% ethanol (carefully avoiding touching the eyes of the animal) and decapitate the animal using large sharp scissors, according to the approved procedure. 
	NOTE: The cerebellar slices are generated from P9-P10 mice without any specific sex or strain bias.</li>
	<li>Use small scissors to cut the skin along the midline of the head starting from the neck and going up to the mouse muzzle.</li>
	<li>To hold the head and expose the skull, fold back the skin ventrally under the head, and pinch the two folds of skin between one&#8217;s fingers.</li>
	<li>Insert the 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. Keep the tip of the scissors as parallel and close to the skull as possible while cutting, to avoid damaging the brain tissue.</li>
	<li>Retrieve the dorsal part of the skull using fine-straight forceps. While carefully lifting the dorsal part of the skull, cut the adhering meninges (translucent irrigated membrane surrounding the brain tissue) if needed with small scissors to avoid damaging the brain tissue.</li>
	<li>Carefully introduce fine-straight forceps (or alternatively use a spatula) between the ventral skull and the brain, gently flip out the brain and cut the optic and trigeminal nerves with small scissors.</li>
	<li>Help the brain to drop by gravity into the 60 mm cell culture dish containing ice-cold dissection medium by turning the head upside down just above the dish and release the last adhesions with small scissors if needed.</li>
	<li>Using fine forceps, orientate the brain with the dorsal side facing the researcher, and the ventral side lying on the bottom of the dish.</li>
	<li>Under the binocular microscope, use the fine-straight forceps to immobilize the brain on the forebrain side and avoid touching the hindbrain, as it could get damaged. Separate the hindbrain from the rest of the brain (fore- and midbrain, see schematic on Figure 1Aa), using fine-straight forceps. To separate the cerebellum from the rest of the hindbrain, use the fine forceps to cut the cerebellar peduncles underneath the cerebellum (see schematic on Figure 1Aa&#8217;).</li>
	<li>Once the cerebellum is isolated, carefully tear away the meninges using fine-straight forceps (see schematic on Figure 1Aa&#8217;).</li>
	<li>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 (see schematic on Figure 1Bb).</li>
	<li>Aspirate any excess of dissection medium around the cerebellum using a sterile thin-end pipette tip adapted on a 1 mL pipette (see schematic on Figure 1Bb).</li>
	<li>Ensure that the cerebellum lays flat, but is not stretched, once placed onto the platform to get optimal sagittal slices during sectioning (see schematic on Figure 1Bb).</li>
	<li>Slice 300 &#956;m-thick sagittal sections of the cerebellum with the tissue chopper (see schematic on Figure 1Bb&#8217;). The force and speed of the blade have to be optimized on site.</li>
	<li>Gently add a drop of dissection medium onto the sliced cerebellum. Using a wide-bore pipette tip placed onto a 1 mL pipette, slowly aspirate the sliced cerebellum and transfer it back into the 60 mm cell culture dish containing ice-cold dissection medium (see schematic on Figure 1Bb&#8217;&#8217;).</li>
	<li>Clean the tissue chopper&#8217;s plastic platform with 100% ethanol between each cerebellum slicing. Let the ethanol evaporate before placing the next cerebellum onto the platform.</li>
	<li>Use two fine-straight forceps (or alternatively titanium needles) to separate individual slices and select 6-8 slices from the vermis (see schematic on Figure 1Cc and 1Cc&#8217;).</li>
	<li>Take a 6-well plate with culture inserts and transfer up to 4 selected slices onto one culture insert along with some dissection medium, using a wide-bore pipette tip adapted on a 1 mL pipette (see schematic on Figure 1Cc&#8217;).</li>
	<li>Place the slices in the middle of the culture insert using the dissection medium or forceps to push and pull them gently into the right location, avoiding them to be in contact with each other (see schematic on Figure 1Cc&#8217;).</li>
	<li>Remove any excess of dissection medium around the slices using a thin-end pipette tip. Ensure that the slices lay flat on the membrane (see schematic on Figure 1Cc&#8217;).</li>
	<li>Place the 6-well plate into the incubator immediately after having added the slices on the membrane.</li>
</ol>
5. Slices Culture and Demyelination (Hands-on Time &#8776; 15&#8211;20 min)
NOTE: Perform this step in a flow culture hood under sterile conditions
<ol>
	<li>Replace the culture medium with 1 mL per well of pre-warmed and buffered fresh culture medium every 3 days after slice preparation.</li>
	<li>For demyelination, remove all culture medium below the culture inserts after 6 days in vitro (DIV) and replace it with 1 mL per well of pre-warmed fresh culture medium containing 0.5 mg/mL LPC. Incubate for 15-17 h at 37 &#176;C, 5% CO2.</li>
	<li>After incubation, wash the inserts by placing them in a 25 mm petri dish containing 1 mL of pre-warmed culture medium. 
	NOTE: The inserts should be in contact with the medium, but not be covered by it.</li>
	<li>Immediately transfer the culture inserts in a new 6-well plate containing fresh pre-warmed culture medium.</li>
	<li>Replace the culture medium with 1 mL of pre-warmed fresh culture medium every 2-3 days until slices fixation.</li>
</ol>
6. Immunohistochemistry (Hands-on Time &#8776; 1:30&#8211;2 h)
NOTE: Carry out the steps 6.1. to 6.3. under a fume hood. Avoid exposition to paraformaldehyde (PFA) and refer to the product safety datasheet for adequate manipulation and protection.
<ol>
	<li>Use forceps to lift each culture inserts by holding the plastic edge and remove the culture medium beneath the membrane inserts from each well.</li>
	<li>Fix the cerebellar slices for 30 min by adding 2 mL of 4% PFA in 1x PBS pH 7.4 onto the membrane inserts. 
	NOTE: The time point of fixation depends on the stage of myelination studied: 4 DIV corresponds to the onset of myelination, 7 DIV to fully myelinated slices. In case of LPC treatment, 9 DIV corresponds to the peak of demyelination, 11 DIV to the onset of remyelination and 14 DIV to fully remyelinated slices.</li>
	<li>Wash the inserts with the slices three times with 2 mL 1x PBS for 10 min.</li>
	<li>Separate the slices from the membrane inserts under the binocular microscope using a 25x to 30x magnification, by gently pushing them using a scalpel or a brush. Alternatively, to avoid the risk of damaging the slices while detaching them from the membrane, use a scalpel to cut the membrane with the slices still attached.</li>
	<li>Transfer the floating slices into the wells of a 4-well plate containing 1x PBS, using a brush.</li>
	<li>Aspirate the PBS from each well and incubate the slices in pre-cooled 100% ethanol at -20 &#176;C for 15 to 20 min. This step facilitates antibody penetration in myelinated fibers.</li>
	<li>Aspirate the 100% ethanol and wash once briefly with 1x PBS, then twice in 1x PBS for 10 min at room temperature.</li>
	<li>Aspirate the PBS and incubate the slices for 30 to 45 min with a solution containing 1x PBS, 5% NGS, 0.3% non-ionic detergent at room temperature to block non-specific antibody fixation sites.</li>
	<li>Aspirate the blocking solution. No washing is necessary.</li>
	<li>Incubate the slices overnight with the primary antibodies diluted in blocking solution at 4 &#176;C. To visualize the myelin on cerebellar organotypic slices, use MBP (Chicken, 1/150 or Mouse IGg2b, Smi99, 1/200) or PLP (Rat, hybridoma, 1/5 to 1/10) antibodies. 
	NOTE: To reveal the expression of more than one antigen in the same slice, perform a double- or triple staining procedure (see Figure 1 as an example). To perform the incubation simultaneously, ensure primary antibodies are produced in different species. Then, use their corresponding secondary antibodies coupled to different fluorophores (see Table of Materials for nodal and paranodal marker18 antibodies).</li>
	<li>On the second day, wash the slices three times in 1x PBS for 10 min.</li>
	<li>Incubate for 3h with the secondary antibodies diluted in blocking solution (1/500 dilution) in the dark at room temperature. 
	NOTE: Optimization of antibody dilution and incubation time might be required when using other antibodies than the ones described here.</li>
	<li>Wash the slices three times in 1x PBS for 10 min in the dark.</li>
	<li>Under a binocular microscope, mount the slices onto a glass slide. Place 100 &#181;L of 1x PBS on the slide and transfer the slices into the PBS using a brush. Unfold the slices if needed and flatten them on the slide. Remove any excess of PBS. In case the immunolabeling is performed with the slices still attached to membranes, mount with the slices facing the coverslip, with the membrane on the glass slide.</li>
	<li>Place a drop of mounting medium directly onto the glass coverslip and gently cover the slices. Mounted slides can be stored for several months at 4 &#176;C in the dark.</li>
</ol>

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None of the authors have competing interests or conflicting interests.

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 cultures taken from 10-day-old mice are a well-established experimental model to investigate molecular and cellular mechanisms underlying myelination and remyelination processes, as it allows to reproduce most anatomical and functional features of the corresponding tissue in vivo including not only the preservation of a well-defined cellular architecture but also the maturation timeline17. Furthermore, cerebellar slices have a preserved architectural organization after two weeks in vitro&#160;(Figure 1C). The transgenic PLP-GFP strain is an alternative, allowing, in particular, to observe the myelination status under a fluorescent binocular microscope on live slices, which is particularly convenient for demyelination-remyelination studies (Figure 1C).
These preparations are of particular interest as a fast approach to assess myelination rate with the developed automated quantification, including western-blot analysis20, CNPase assay16 or quantification of PLP/Calbindin (or MBP/Calbindin) staining17,21, thus representing a high-throughput system allowing for the testing of pharmacological drugs in demyelinating disorders22,23.
One of the main critical issues when performing these experiments might be related to the quality of the cerebellar slices. Firstly, the age of mice is of importance for cerebellar organotypic slice cultures. The survival of Purkinje cells, the only myelinated neuron in cerebellar cultures, is compromised if cultures are prepared from P2 to P7 rodents or with donors older than P1317. More generally, organotypic brain slice cultures are difficult to obtain from adults animals. Secondly, the duration of tissue processing during dissection is critical, as a dissection step of more than 15-20 minutes per animal leads to a decline in the survival rate of the slices. Furthermore, when selecting the slices to be cultured, it is best to avoid using the ones from either end of the cerebellum due to the poor survival of neurons and resultant scarcity of myelinated fibers.
Other parameters such as the stability of temperature and CO2 concentration while in culture are determining factors to ensure slice health. Lastly, the quality of the slices is influenced by the composition of the horse serum, which cannot be controlled by the users. Therefore, testing several batches of serum is highly recommended.
To obtain an adequate labeling, tissue fixation is a key step. In order to facilitate the penetration of the antibodies to reach myelin antigens, as myelin is a very compact structure, slices pre-treatment with absolute ethanol is advised following fixation. A similar protocol of staining can further be applied to in vivo fixed tissues, though Triton concentration may need to be adapted to the thickness of vibratome or cryostat generated in vivo sections. The availability of non-commercial antibodies should also be considered. Preparing slices from transgenic mice such as PLP-GFP24 or CNP-GFP25 thus represents an alternative to visualize myelin and oligodendrocytes processes.
In addition to the immunochemical methods described in this protocol, other approaches are commonly used to study myelin architecture. Complementary techniques also exist to investigate myelin ultrastructure, with electron microscopy being the gold standard26. Other techniques exist, such as label-free approaches including optical coherence tomography27,28, Raman scattering28,29, third-harmonic generation30 or spectral confocal reflectance microscopy31; these approaches have the additional advantage of enabling the observation of dynamic cellular processes. However, some of these techniques are complex and further require state-of-the-art microscopy set-ups. For these reasons, myelin study by immunofluorescence labeling remains a standard approach to investigate myelination and its defects in various developmental or acquired diseases and their models, as well as to assess the potential of future therapeutic treatments. It can further be used to study myelination in multiple contexts, such as learning and plasticity, as well as aging, depression, or autism in which myelin is known to be altered32.

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&amp;rsquo;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&amp;ndash;Streptomycin (10.000 IU/mL)</td><td>ThermoFisher Scientific</td><td>15140122</td><td></td></tr><tr><td>Gey&amp;rsquo;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&amp;deg;C</td></tr><tr><td>Triton&amp;reg; 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 &amp;micro;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&amp;deg;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 &amp;micro;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 mm x 26 mm x 1.1 mm</td></tr><tr><td>Glass coverslips</td><td>RS France</td><td></td><td>22 mm 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 induction of a full demyelination is possible through LPC treatment (Figure 2Ci-ii). Following demyelination, the slices spontaneously remyelinate and are fully remyelinated 6-7 days after the peak of demyelination (Figure 2Ciii).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59163/59163fig1.jpg" /> 
Figure 1: Illustration of cerebellum slices generation. The dissection is divided in three main steps. (A) The cerebellum is isolated and the meninges removed (steps 4.10 and 4.11). (B) The cerebellum is then transferred to the chopper platform and sliced (steps 4.12 to 4.16). (C) Lastly, the slices are isolated from the vermis and placed on a membrane insert. The dissection medium transferred with the slices is removed and the 6-well plate is placed into the incubator (steps 4.18 and 4.22). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59163/59163fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59163/59163fig2.jpg" /> 
Figure 2: Example of myelinated cerebellar slices cultured ex vivo. Image stacks (with orthogonal projection) were obtained using an upright confocal microscope following free floating immunolabeling and flat mounting as described in the protocol. The slices are robustly myelinated at 11 DIV (A, PLP; B, GFP in green)&#160;and the axonal domains are assembled as observed in vivo with nodes of Ranvier enriched in voltage-gated sodium channels (Nav, in red) flanked by the paranodal axoglial junction domain (Caspr, in white) in C57black6 WT (A) as well as PLP-GFP (B) transgenic mice slices. (C) The cerebellar cortex architecture is preserved in the cultured slices, as observed on cerebellar slices from PLP-GFP mice. (i) Purkinje cells axon (Calbindin, Calb, in blue) are robustly myelinated (GFP, in green) after one week in culture. (ii) LPC treatment fully demyelinates the slices, which remyelinate spontaneously and are fully remyelinated 6 days post demyelination (iii, 14 DIV). Scale bars = 20 &#181;m (A, B); 100 &#181;m (C). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59163/59163fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

preparation-immunostaining-myelinating-organotypic-cerebellar-slice

Nanyang Technological University

Research

JoVE Journal

Neuroscience

10.9K Views.  Sorbonne Université, Inserm, CNRS, Institut du Cerveau et de la Moelle épinière, ICM-GH Pitié-Salpétrière. 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.

Video: Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

Developmental Biology

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced tools currently used to study neurons, have only partly been taken on by oligodendrocyte researchers. Cell type-specific staining by viral transduction is a useful approach to study live organelle dynamics. This paper describes a protocol for visualizing oligodendrocyte mitochondria in organotypic brain slices by transduction with adeno-associated virus (AAV) carrying genes for mitochondrial targeted fluorescent proteins under the transcriptional control of the myelin basic protein promoter. It includes the protocol for making organotypic coronal mouse brain slices. A procedure for time-lapse imaging of mitochondria then follows. These methods can be transferred to other organelles and may be particularly useful for studying organelles in the myelin sheath. Finally, we describe a readily available technique for visualization of unstained myelin in living slices by Confocal Reflectance microscopy (CoRe). CoRe requires no extra equipment and can be useful to identify the myelin sheath during live imaging.

Myelinating oligodendrocytes promote rapid action potential propagation and neuronal survival. Described here is a protocol for oligodendrocyte-specific expression of fluorescent proteins in organotypic brain slices with subsequent time-lapse imaging. Further, a simple procedure for visualizing unstained myelin is presented.

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced ...

Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early postnatal development, cerebellar Purkinje cells are known to go through a vulnerable period, during which they undergo programmed cell death. Here, we provide a detailed protocol to perform mouse organotypic cerebellar slice culture during this critical time. The slices are further labeled to assess Purkinje cell survival and the efficacy of neuroprotective treatments. This method can be extremely valuable to screen for new neuroactive molecules.

Organotypic slice cultures are a powerful tool to study neurodevelopmental or degenerative/regenerative processes. Here, we describe a protocol that models the neurodevelopmental death of Purkinje cells in mouse cerebellar slice cultures. This method may benefit research in neuroprotective drug discovery.

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early ...

Ex Vivo Myelination and Remyelination in Cerebellar Slice Cultures as a Quantitative Model for Developmental and Disease-Relevant Manipulations

Studying myelination&#160;in vitro&#160;and&#160;in vivo poses numerous challenges. The differentiation of oligodendrocyte precursor cells (OPCs)&#160;in vitro, although scalable,&#160;does not recapitulate axonal myelination. OPC-neuron cocultures and OPC-fiber cultures allow for the examination of in vitro myelination, but they lack additional cell types that are present in vivo, such as astrocytes and microglia. In vivo&#160;mouse models, however, are less amenable to chemical, environmental, and genetic manipulation and are much more labor intensive. Here, we describe&#160;an&#160;ex vivo mouse cerebellar slice culture (CSC) quantitative system that is useful for: 1) studying developmental myelination, 2) modeling demyelination and remyelination, and 3) conducting translational research. Sagittal sections of the cerebellum and hindbrain are isolated from postnatal day (P) 0&#8211;2 mice, after which they myelinate&#160;ex vivo&#160;for 12 days. During this period, slices can be manipulated in various ways, including the addition of compounds to test for an effect on developmental myelination. In addition, tissue can be fixed for electron microscopy to assess myelin ultrastructure and compaction. To model disease, CSC can be subjected to acute hypoxia to induce hypomyelination. Demyelination in these explants can also be induced by lysolecithin, which allows for the identification of factors that promote remyelination. Aside from chemical and environmental modifications, CSC can be isolated from transgenic mice and are responsive to genetic manipulation induced with Ad-Cre adenoviruses and tamoxifen. Thus, cerebellar slice cultures are a fast, reproducible, and quantifiable model for recapitulating myelination.

Presented is a protocol for an ex vivo quantitative model of demyelination and remyelination using mouse cerebellar slice cultures. This method closely recapitulates an in vivo model with its full complement of CNS cell types in intact tissue, while maintaining the chemical, genetic, and environmental amenability of an in vitro system.

Studying myelination&#160;in vitro&#160;and&#160;in vivo poses numerous challenges. The differentiation of oligodendrocyte precursor cells (OPCs)&#160;in ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

Biology

Cerebellum Dissection and Slice Preparation: A Method to Generate Tissue Slices from Mouse Cerebellum

Source: Thetiot, M. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/59163" target="_blank">Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures.</a> J. Vis. Exp. (2019)
This video describes a method to generate tissue slices from the mouse cerebellum. The cerebellar slices can be used for organotypic culturing that provides insights into neurodevelopmental mechanisms, including myelination dynamics.

Rozwarstwienie móżdżku i przygotowanie plastrów: metoda generowania wycinków tkanek z móżdżku myszy

Source: Thetiot, M. et al. Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures. J. Vis. Exp. (2019)
This video describes a ...

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

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Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy

Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

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