Name: Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection (Video) | JoVE Journal
Uploaded: 2011-05-17T12:00:00.000+00:00
Duration: 11 min 57 s
Description: Learn organotypic cerebellar culture techniques to study apoptotic challenges in wild type and mutant mice using immunofluorescent staining. This method enables detailed analysis of cell viability and death pathways.

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This study was funded in part by the IPSEN Foundation. IMV is an American Cancer Society Professor of Molecular Biology, and holds the Irwin and Joan Jacobs Chair in Exemplary Life Sciences. This work was supported in part by grants from the NIH, Leducq Foundation, Meriaux Foundation, Ellison Medical Foundation, Ipsen/Biomeasure, Sanofi Aventis, Prostate Cancer Foundation, and the H.N. and Frances C. Berger Foundation. PAS is funded by McKnight Endowment Fund for Neuroscience and the National Institute on Drug Abuse (R01 DA019022). The authors would like to thank Mark Schmitt. Graphic art was designed by Jamie T. Simon .Sponsorship was kindly provided by Invitrogen.

1. Organotypic Cerebellar Cultures:
<ol>
<li>To successfully generate organotypic cerebellar cultures use mice between P0-P3 or P8-P12. For our experiment we used mice in the P8-P12 range because this developmental stage was more appropriate for our study.</li>
<li>Prepare the dissection and the culture medium:
<ol>
<li>Brain dissection and slice preparation are carried out in low sodium Artificial Cerebrospinal Fluid (ACSF) containing 1mM calcium chloride, 10mM D-Glucose, 4mM potassium chloride, 5mM magnesium chloride, 26mM sodium bicarbonate, 246mM sucrose and phenol red solution (1:1000), pH 7.3. To sterilize the solution, filter it through a .22 μm filter and store at 4 °C.</li>
<li>Cerebellar slices are cultured in 75% Minimum Essential Medium Eagle (MEM), 25% heat-inactivated horse serum, 25mM HEPES, 1mM glutamine, 5mg/ mL glucose, penicillin (100 U/ mL) and streptomycin (100 U/ mL). Filter the medium through a 0.22 μm filter and store at 4 °C for a maximum of two weeks.</li>
</ol>
</li>
<li>Dissect brain in ice-cold ACSF medium previously bubbled with Carbogen (95% oxygen and 5% carbon dioxide) Note, that saturation with Carbogen will change the pH of the solution so it will shift from red to orange. The dissection should be performed in the minimum amount of time possible being careful not to damage the brain structure.</li>
<li>Slice the brain in sagittal sections of 400 μm using a vibratome.
<ol>
<li>Make a sagittal cut with a razor blade in one hemisphere, trying to cut as little as possible of the cerebellum; this will provide a flat surface for slicing. The cortex is used only for support to make the slicing easier but since time is an important issue it is more convenient to cut out the rostral half of the cortex with a razor blade.</li>
<li>Use cyanoacrylate (Superglue) to fix the position of the brain on the metal vibratome plate.
It is important to chill the plate on ice before adding the glue and to spread it well using the tip of the tube to form a thin adhesive layer. If there is an excess of Superglue, it will detach from the plate when it comes in contact with the ACSF and the sample will be lost.</li>
<li>Once the brain is attached to the vibratome plate, add enough ACSF media to cover the brain and put ice below the plate to keep it cold.</li>
<li>Cut 400 μm slices and transfer them with a plastic Pasteur pipette (cut the tip to widen it and avoid damage to the slices) to a 6-well cell culture plate containing ACSF on ice.</li>
<li>Separate the cerebellum from the rest of the brain with the help of two insulin syringes (syringes with small diameter 28-gauge needles) under the dissecting microscope. Some slices will have Superglue attached to the edges: this is the moment to try to remove it as it can affect the viability of the slice. It is important to do it carefully without damaging the cerebellar structure.</li>
</ol>
</li>
<li>Transfer the cerebellar slices to 6-well plates containing 30mm culture plate inserts with 0.4 μm pores. Add 1 mL of culture media per well, that was pre-conditioned by incubation for at least 2 hours at 37°C and 5% CO2 (in the cell culture incubator). Place the inserts on top of the media making sure there are no air bubbles trapped underneath and then place the cerebellar slices (1-3 per insert) on top of the inserts making sure they are fully extended and that no liquid is covering them. Aspirate excess of media if necessary. Incubate at 37°C and 5% CO2 changing the culture media every 2 or 3 days. It is best to replace only a portion of the medium (e.g. ½ volume) at once, since used medium contains slice-derived trophic factors that are important for cell survival.</li>
</ol>
2. Fas Treatment and Staining of Cerebellar Slices:
<ol>
<li>For the apoptotic challenge experiment, we first culture the slices for 5 days changing media on day in vitro 3 (DIV3). On DIV5 cultures are treated with 0.5 μg/ mL of the Fas agonist antibody Jo2 by adding it directly to the media in order to have a clean background of cell death since the changes of media can induce some mortality in the cultures. Half of the wells are left untreated as controls.</li>
<li>After 24 hours remove the media underneath the inserts by suction. To fix the slices, add 1 mL of ice cold 4% PFA beneath and 1 mL above the insert. Incubate for 10 minutes at room temperature.</li>
<li>After 10 minutes remove the PFA and wash briefly with cold PBS.</li>
<li>Next remove the PBS and add 1 mL beneath and 1 mL above the insert of 20% ice-cold methanol in PBS and incubate for 5 minutes at room temperature.</li>
<li>Wash again with PBS and add 1 mL beneath and 1 mL above the insert of 0.5% Triton-X-100 in PBS for permeabilization. Incubate for a minimum of 12 hours at 4°C.</li>
<li>After permeabilization, block with 20% BSA in PBS for a minimum of 4 hours at room temperature or overnight at 4°C. At this stage the slices can be kept in blocking solution for at least 3 days at 4°C.</li>
<li>In order to stain the slices carefully cut the piece of the membrane insert attached to the cerebellar slice and transfer it to 24-well plates containing PBS.</li>
<li>The immunostaining is done sequentially, first with TUNEL reagent for one hour and then with Calbidin antibody overnight.
<ol>
<li>Aspirate the PBS and add 250 μL off TUNEL reagent into each well making sure it covers the entire surface of the slice. Incubate at 37°C for one hour in the dark.</li>
<li>After one hour remove the TUNEL mix and wash with PBS three times for 10 minutes.</li>
<li>After the last wash, remove the PBS and add 250 μL of a 1/1000 dilution of the Calbindin antibody in PBS and incubate overnight at 4°C in the dark.</li>
<li>Remove the primary antibody solution and wash with PBS three times for 10 minutes.</li>
<li>Add 250 μL of secondary antibody diluted 1/500 in PBS and incubate for at least three hours at room temperature in the dark. The TUNEL kit we used is labeled with TMR red, therefore our secondary antibody for Calbindin is conjugated to Alexa-488.</li>
<li>Wash three times for 10 minutes with PBS and mount the slices onto a glass microscope slide using a mounting media that contains DAPI.</li>
<li>Image the slices with a confocal microscope using the 488 nm and 561 nm excitation wavelenght for Calbindin and TUNEL respectively.</li>
</ol>
</li>
</ol>
3. Representative Results:
The main challenge of this protocol is to be able to generate healthy cerebellar slice cultures, especially since our final readout will be cell death. A healthy culture appears as one where the cell body layer is translucent and allows you to see the foliated structure of the cerebellum under the microscope (Figure 1). After few days in culture the slices start to thin and non-neuronal cells can be observed migrating away from the margins of the slice. The dark round cells (most likely macrophages) that cover the slices after few days in culture, will eventually decrease in number after 10-14 days in vitro (Figure 2) 5. The ultimate proof for the viability of the slices is to stain for different cellular markers, such as Calbindin for the Purkinje cells. Figure 3 shows a representative confocal microscope image of the Purkinje cell layer with several TUNEL positive nuclei.
It is important to consider that even if some slices received an apoptotic stimulus, they were only exposed for 24 hours. This allowed sufficient time to observe DNA fragmentation in the dying cells with the TUNEL staining, but a defined Purkinje cell layer was still present. 
<img src="/files/ftp_upload/2564/2564fig1.jpg" alt="Bacterial colony morphology diagram, petri dish, microbial growth, biological research." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. Healthy cerebellar slice at DIV2. This image shows the translucent cell body layer and the foliated cerebellar structure in a healthy slice.
<img src="/files/ftp_upload/2564/2564fig2.jpg" alt="Embryonic development, microscope image, early frog embryo, cellular differentiation, gastrulation." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. Healthy cerebellar slice at DIV 7. In this image we can still observe the foliated structure of the cerebellum and the appearance of dark cell bodies.
<img src="/files/ftp_upload/2564/2564fig3.jpg" alt="Microscopy analysis, neural tissue fluorescence, cell visualization, microscope image, neurobiology study." data-altupdatedat="1788235205000" data-alt="Figure 3"> 
Figure 3. Representative image of a Fas-treated cerebellar slice. This confocal image shows the Purkinje cells stained with Calbindin (green) and the apoptotic cells positive for TUNEL staining (red). Purkinje cells do not appear in a single row but are clustered forming a folium-like structure.

<ol><li>Hogue, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+fetal+ependymal+cells+in+tissue+cultures%5BTitle%5D)+AND+%22Anat+Rec%22%5BJournal%5D)">Human fetal ependymal cells in tissue cultures.</a> Anat Rec. 99, 523-529 (1947).</li>
<li>Hogue, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Review+of+studies+of+human+fetal+brain+cells+in+tissue+cultures%5BTitle%5D)+AND+%22Etudes+Neonatales%22%5BJournal%5D)">Review of studies of human fetal brain cells in tissue cultures.</a> Etudes Neonatales. 1, 1-13 (1952).</li>
<li>Gogolla, N., Galimberti, I., DePaola, V., Caroni, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Staining+protocol+for+organotypic+hippocampal+slice+cultures%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Staining protocol for organotypic hippocampal slice cultures.</a> Nat Protoc. 1, 2452-2456 (2006).</li>
<li>Stoppini, L., Buchs, P. A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+simple+method+for+organotypic+cultures+of+nervous+tissue%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">A simple method for organotypic cultures of nervous tissue.</a> J Neurosci Methods. 37, 173-182 (1991).</li>
<li>Beat, H., Gähwiler, S. M. T., McKinney, R. A., Debanne, D., Robertson, R. T. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBeat%2C+H+%22Organotypic+Slice+Cultures+of+Neural+Tissue+in+Culturing+Nerve+Cells%22">Organotypic Slice Cultures of Neural Tissue in Culturing Nerve Cells</a>. Banker, G., Goslin, K. , MIT Press. Cambridge. 461-498 (1998).</li>
</ol>

All animal work conducted was done in accordance with policies established by the IACUC. The Salk Institute is an AAALAC accredited facility.

This method describes one of the many possible applications of neuronal organotypic cultures and it has allowed us to study the effect of apoptotic stimuli in wild type and mutant cerebellar slices.
The most critical part of this experiment is to be able to consistently generate healthy cerebellar slice cultures. The key factors are dissection and slicing technique and the ability to perform the protocol in the least time possible, but at the same time being careful not to damage the slices. Another crucial factor is the preparation and composition of the culture media, these cultures are very sensitive, so we had to test several recipes and brands of media. Even different batches of serum or any other components of the media could affect the viability of the slices. We got the best results with the Horse Serum from Invitrogen Lot # 480116.
Once the cerebellar slices are generated one can study cerebellar development, electrophysiology, cell survival alone or in response to apoptotic stimuli, excitotoxicity or oxygen deprivation. Comparative studies using wild type and mutant cerebellar slices have been very insightful in many aspects of cerebellar function and development.

<table><tbody><tr><td>MEM</td><td>Invitrogen</td><td>11090</td><td></td></tr><tr><td>Horse Serum</td><td>Invitrogen</td><td>16050</td><td></td></tr><tr><td>Hepes</td><td>Invitrogen</td><td>15630</td><td></td></tr><tr><td>Glutamine</td><td>Invitrogen</td><td>25030</td><td></td></tr><tr><td>Penicillin/ Streptomycin</td><td>Invitrogen</td><td>15140</td><td></td></tr><tr><td>Superglue</td><td>Krazy Glue</td><td></td><td></td></tr><tr><td>6 well / 24 well cell culture plates</td><td>Corning</td><td>3506/ 3527</td><td></td></tr><tr><td>30mm culture plate inserts (0.4-&amp;mu;m pore)</td><td>EMD Millipore</td><td>PICM03050</td><td></td></tr><tr><td>Purified NA/LE Hamster anti-mouse CD95 (Jo2)</td><td>BD Biosciences</td><td>554254</td><td></td></tr><tr><td>&lt;em&gt;In situ&lt;/em&gt; cell death detection kit, TMR red</td><td>Roche Group</td><td>12 156 792 910</td><td></td></tr><tr><td>Rabbit anti-Calbindin D-28k antibody</td><td>Swant</td><td>CB-38a</td><td></td></tr><tr><td>Alexa 488-goat anti-rabbit IgG secondary antibody</td><td>Invitrogen</td><td>A-11008</td><td></td></tr><tr><td>Vectashield Mounting Medium with DAPI</td><td>Vector Laboratories</td><td>H-1200</td><td></td></tr><tr><td>Dissecting Microscope</td><td>Carl Zeiss, Inc.</td><td></td><td></td></tr><tr><td>Vibratome</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Confocal Microscope</td><td>Leica Microsystems</td><td></td><td></td></tr></tbody></table>

organotypic cerebellar cultures: apoptotic challenges and detection

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 overall goal of the following experiment is to investigate the effect of the apoptotic stimulus fast ligand in the developing cerebellum of wild type and mutant mice. This is accomplished by first dissecting the brain of wild type and knockout mice between postnatal days eight and 12. The second step is to slice the brains in 400 micrometer thick slices and isolate the cerebellum from each slice.The third step is to culture the cerebellar slices for five days to make sure they are healthy before inducing cell death with fast ligand. The final step of the procedure is to analyze the effect of fast ligand in the different cerebellar cell populations of wild type and knockout mice based on immunofluorescent staining for the apoptotic marker tunnel, combined with cell type specific markers like calbindin for bikini cells. The main advantage of cerebellar life cultures is that it allows the style of cerebellar regional structure and like primary dissociated cultures where the neuronal networks and several organizations are disrupted.So this matter will allow to answer some key questions in neurosciences, especially because one has the wild type and the mutant mouse. So one can study comparative development, one can study physiology, and also electrophysiology and pharmacology. Though this method can provide insights into the function of fast ligand in cerebellar or cell survival, it can also be used inside of the effects of fast ligand in cortex and hippocampus.Generally, people new to this method will struggle because in order to obtain healthy cerebellar lysis, it is critical to put them into culture as quickly as possible without compromising their integrity. Therefore, it's important to have a good dissection and slicing skills before attempting the whole process. Where possible.The steps of the following procedure should be performed in a tissue culture hood prior to dissection. Prepare A CSF media for brain dissection and slice preparation, as well as slice culture media according to the written protocol and store them at four degrees Celsius on the day of dissection. Prepare tissue culture plates for culturing the cerebellar slices.Add one milliliter of slice culture media to each well of a six well plate. Then use sterile forceps to place the culture plate inserts into each. Well ensure that there are no air bubbles trapped beneath the insert.Place the culture plate in a tissue culture incubator Set at 37 degrees Celsius and 5%carbon dioxide for at least two hours to precondition the media. Next, bubble the ice cold. A CSF media with carbogen gas saturation of the media with carbogen alters the pH of the solution and produces a color change from red to orange.Pour a little of the ice cold bubbled A CSF media into a sterile Petri dish and a six well plate. Place these on ice together with the plate of the vibrato that will be used for slicing the brain. The Petri dish containing bubbled ice cold A CSF media will be used for dissecting the brain from a P eight to P 12 mouse pup following dissection.Use a razor blade to make a sagittal cut in one hemisphere to provide a flat surface for mounting on the vibram plate. The rostral part of the cortex may also be cut. Dry the chilled vibram plate with a paper towel.Then add a little super glue to the stage and spread it in a zigzag manner with the tip of the super glue tube. To form a thin adhesive layer, use a spatula to transfer the brain to the vibrato stage with the flattened side in contact with the super glue. Once the brain is attached to the vibram plate, add enough A CSF media to cover the brain and add ice below the plate to keep it cold.Cut the brain into 400 micrometer slices. Then use a plastic pasta pipette with a cut. Widened end to transfer the slices to the six well plate containing A CSF media on ice working under the dissecting microscope.Use two insulin syringes with small diameter 28 gauge needles to separate the cerebellum from the rest of the brain. If any of the slices have super glue on the edge, use the two syringe needles to remove the glue from the slice. Use the plastic pasta pipette with a cut widened end to transfer the slices onto the top of the filters.Adding one to three slices per insert ensure that the slices are fully extended and that no liquid is covering them. If necessary, aspirate excess media with the pipette incubated, 37 degrees Celsius and 5%carbon dioxide for 48 hours. After 48 hours, replace half of the media volume with fresh media, making sure that no air bubbles are trapped underneath the insert.The remaining original medium contains sliced derived trophic factors that are important for cell survival. The apoptotic challenge is performed after five days of culture. Add the fast agonistic antibody JO two to the cell culture media in the wells to be treated.Leave half of the wells untreated as controls. Once 24 hours have elapsed, aspirate the media from under the wells. Then fix the slices by pipetting one milliliter of ice cold, 4%paraldehyde under the insert and one milliliter of PFA on top of the insert.Allow the slices to fix in the PFA, the 10 minutes of room temperature after 10 minutes. Aspirate the PFA and wash briefly with two milliliters of cold PBS pipetting. One milliliter on top of the insert and one milliliter below the insert.Next, aspirate the PBS and pipette one milliliter of 20%ice cold methanol in PBS underneath the insert and one milliliter above the insert. Incubate for five minutes of room temperature, then aspirate the 20%methanol from the well and wash the slice and D well with PBS. As before, pipette one milliliter of 0.5%Triton X 100 in PBS on top of the insert, and another one milliliter underneath the insert.Incubate for a minimum of 12 hours at four degrees Celsius to perme the slices following permeation. Aspirate the 0.5%Triton X 100 in PBS and pipette one milliliter of 20%BSA in PBS on top of the slice and one milliliter beneath the slice. Block the slices for a minimum of four hours at room temperature or overnight at four degrees Celsius.When blocking is complete, use clean scissors to carefully cut the piece of membrane insert attached to the cerebellar slice. Then use clean forceps to transfer the piece of membrane into a 24 well plate containing PBS aspirate the PBS and add 250 microliters of tunnel reagent into each well. Making sure that the reagent covers the entire surface of the slice.Incubate at 37 degrees Celsius for one hour in the dark when the hour has elapsed. Aspirate the tunnel reagent from the wells and replace with 500 microliters of PBS. Protect the plate from light and incubate it room temperature for 10 minutes to wash.Repeat this process twice after the last wash. Aspirate the PBS and add 250 microliters of a one to 1000 dilution of the calbindin antibody in PBS and incubate overnight at four degrees Celsius in the dark. The next day.Aspirate the primary antibody solution and wash with PB S3 times for 10 minutes. Then add 250 microliters of secondary antibody diluted one to 500 in PBS. Cover the plate with aluminum foil to protect from light and incubate for at least three hours of room temperature following incubation.Wash the slices three times for 10 minutes with PBS and using mounting media that contains dappy. Mount the slices onto a glass microscope, slide and cover slip. Image the slices with a confocal microscope using the 488 nanometer and 561 nanometer excitation wavelengths for kale, bendin, and tunnel respectively.This image shows a healthy cerebellar slice. After two days in vitro, the cell body layer appears translucent and the foliated structure of the cerebellum is clearly seen under the microscope. After seven days in vitro, the foliated structure of the healthy cerebellar slice is still visible.In addition, dark cell bodies, probably macrophages usually cover the slice. The confocal image seen here is representative of a fast treated cerebellar slice. The bikini cells appear green after staining with a in primary antibody, followed by an Alexa 4 88 conjugated secondary antibody.The apoptotic cells positive for tunnel staining appear red as the tunnel kit used is labeled with TMR. Red Bikini cells do not appear in a single row, but a clustered forming a like structure While attempting this procedure. It is important to remember to work in tissue culture hood whenever possible, and to minimize the amount of time the cerebral tissue is exposed to possible contaminants like bacteria or fungi.Using this procedure, other methods can be applied to these slices, including biochemistry and electrophysiological analyses to provide additional information on protein content as well as neuronal excitability. This technique was originally developed to study the effect of fast mediated cell death in Perkin cells carrying mutations that affect the fast pathway. Perial cells rarely survive in primary culture.So this procedure was the best experimental approach. So I hope after watching this video, you'll have a good idea as to how to make cere barrel slices and how one can study the apathetic stimuli and its effect on different cerebral cell types.

organotypic-cerebellar-cultures-apoptotic-challenges-and-detection

Research

JoVE Journal

Neuroscience

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Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization

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The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

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Analyzing Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

This video demonstrates the method of identifying neurons from a mixed culture of cerebellar granule neurons and glial cells using dual staining with fluorescein diacetate (FDA) and propidium iodide (PI), where live neurons appear green and dead neurons appear red.

Assessing Neuronal Viability in a Cerebellar Neuron Culture via Double Staining

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.

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

This protocol describes how to accurately measure neuronal viability using Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining in cultured cerebellar granule neurons, a primary neuronal culture used as an in vitro model in neuroscience and neuropharmacology research.

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

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.

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

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.

Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

Cerebellar Purkinje cells (PCs) are particularly sensitive to deficiencies in the DNA damage response. A protocol is presented for the visual evaluation of the dynamics of PC response to DNA damage, which involves staining protein-bound poly(ADP-ribose) chains within cerebellar organotypic cultures.

Visualizing the DNA Damage Response in Purkinje Cells Using Cerebellar Organotypic Cultures

Learn organotypic cerebellar culture techniques to study apoptotic challenges in wild type and mutant mice using immunofluorescent staining. This method enables detailed analysis of cell viability and death pathways.

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

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