Work in our lab is supported by the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation and the Israeli Association for Fighting the A-T Disease.

See Table of Materials for details of materials, equipment, and antibodies. Animal procedures were employed under the ethical guidelines of Tel Aviv University&#39;s Ethics Committee after approval. The procedure is carried out on 10-day old mouse pups, regardless of their sex. If needed, genotyping is performed on the previous day using a tail biopsy and standard methods. Solutions are sterile and stored at 4 &#176;C unless indicated otherwise; see Table 1.
<st...

Cerebellar Purkinje Cells: DNA Damage Response in Organotypic Cultures

General comments 
The major advantage of the organotypic culture system is that it facilitates studies using the cerebellar cortex tissue, preserving its structural organization for several weeks in the culture dish setup. This system is useful for conducting in-depth morphological analyses of Purkinje cells (PCs), including detailed examinations of dendritic spines and ultrastructural features52,53,54...

The integrity of cellular DNA is constantly under threat from DNA damaging agents, predominantly metabolic by-products like reactive oxygen species, which inflict tens of thousands of DNA lesions per cell daily1. The persistent upkeep of genome stability is essential for cellular homeostasis2,3. The cornerstone of this maintenance is the DNA damage response (DDR) - an intricate, layered signaling network that initiates specific DNA repair pathways while carefully adjusting many other cellular processes4,5. Deficits in the DDR are commonly manifested as &#39;genome instability syndromes,&#39; marked by chromosomal instability, progressive tissue deterioration, impaired growth or development, a predisposition to cancer, and heightened sensitivity to particular DNA-damaging agents6,7,8,9. Notably, neurodegeneration, which often includes cerebellar atrophy, is a distinct feature of many genome instability syndromes7,10,11,12.
The autosomal recessive disorder, ataxia-telangiectasia (A-T), is a well-documented example of a genome instability disorder13,14,15. This condition arises from null mutations in the ATM (A-T, mutated) gene, responsible for coding the pivotal protein kinase, ATM, which is known primarily as a DDR mobilizer in response to DNA double-strand breaks (DSBs)16,17. A-T manifests as a multisystem disorder, predominantly characterized by progressive cerebellar degeneration, leading to acute motor impairments, immunodeficiency, gonadal atrophy, cancer predisposition, and extreme sensitivity to ionizing radiation. Cultured cells from individuals with A-T show chromosomal instability and increased sensitivity to genotoxic agents, especially those causing DSBs15,18,19. Importantly, ATM also plays a role in repairing other DNA lesions, underscoring its broad significance in maintaining genome stability20,21,22.
Despite thorough research into ATM&#39;s numerous roles, the specific mechanism leading to cerebellar degeneration in A-T remains a topic of active debate, with various models proposed to elucidate this process23,24,25,26,27,28,29,30. Our model28 suggests that cerebellar degeneration in A-T patients begins with the dysfunction and eventual loss of Purkinje cells (PCs). Considering ATM&#39;s critical role in preserving genome stability in the face of ongoing DNA damage, PCs are particularly vulnerable to the absence of ATM. We attribute this vulnerability to the combination of their high metabolic activity, distinctive chromatin structure, and extensive transcriptional activity. Ultimately, it is suggested that the loss of PC function, and hence, their degeneration, is due to the stochastic, functional inactivation of genes, a consequence of producing defective transcripts28.
The study of PC biology in the laboratory is impeded by challenges in cultivating isolated PCs, as these cells rely heavily on their natural milieu and neighboring cells for survival and function, rendering them incompatible with dissociated culture growth. Nonetheless, PCs can remain viable for extended periods in tissue slice cultures. Cerebellar organotypic cultures, which are tissue slices typically derived from rodent cerebella, maintain the tissue&#39;s structural organization and support various experimental manipulations analogous to those possible with cultured cells. Therefore, these cultures allow for cerebellar studies within a controlled setting31,32,33,34,35,36,37,38,39,40,41,42,43. Specifically, in the context of A-T, murine cerebellar organotypic cultures have proven to be instrumental in exploring the DDR in Atm-deficient PCs40,41,42,43. While Atm-deficient mice display only a subtle cerebellar phenotype44,45,46,47, presumably due to differences between human and mouse cerebellar physiology, the assumption is that ATM&#39;s roles are largely conserved across these species. This notion is supported by our observations that the deficient response to DNA DSBs in Atm-/- murine PCs aligns with that observed in other murine and human ATM/Atm-deficient cell types40,41,42,43.
A limitation in analyzing PC responses to various stimuli or stresses within organotypic cultures is the necessity to rely on microscopic imaging for readouts. The DDR is usually studied using bulk biochemical readouts, although common immunofluorescent markers are utilized as well, such as following the dynamics of formation and resolution of nuclear foci of phosphorylated histone H2AX (&#947;H2AX) and the 53BP1 protein, which are considered indicators of DSBs48,49. A broader measure is the fluorescent imaging of poly(ADP-ribose) (PAR) chain formation on proteins, a rapid and robust early DNA damage response, particularly to strand breaks7,50. We modified the protocol by Komulainen et al.51 for PAR staining in cerebellar organotypic cultures. We observed a pronounced PAR response in the sizeable nuclei of PCs. Presented here is our refined protocol for establishing murine cerebellar organotypic cultures and for visualizing the PAR response under genotoxic stress.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent and Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>150 x 30 cm Petri dish</td>
			<td>Corning Inc.</td>
			<td>3261</td>
			<td></td>
		</tr>
		<tr>
			<td>35 x 10 mm Petri dish</td>
			<td>Corning Inc.</td>
			<td>3294</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Invitrogen</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>270725-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive microscope slides</td>
			<td>Marienfeld</td>
			<td>48187</td>
			<td>76 x 26 x 1 mm</td>
		</tr>
		<tr>
			<td>Blades for chopper</td>
			<td>TED PELLA</td>
			<td>Model TC752-1</td>
			<td>&#160;Style Razor Blades</td>
		</tr>
		<tr>
			<td>BME</td>
			<td>GIBCO</td>
			<td>41010-026 500ml.&#160;</td>
			<td>No L-Glut</td>
		</tr>
		<tr>
			<td>Cell culture inserts</td>
			<td>Millipore</td>
			<td>PICM0RG50</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Biological Indo.</td>
			<td>02-015-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS w/o phenol red</td>
			<td>Sigma-Aldrich</td>
			<td>H-1138-500L</td>
			<td>No Ca+,No Mg+</td>
		</tr>
		<tr>
			<td>HBSS with phenol red</td>
			<td>Sartorius</td>
			<td>02-015-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum&#160;</td>
			<td>GIBCO</td>
			<td>26050088</td>
			<td>heat inactivated</td>
		</tr>
		<tr>
			<td>Iris forceps</td>
			<td>CellPath</td>
			<td>GZX-0100-00A</td>
			<td>130mm blunt serrated</td>
		</tr>
		<tr>
			<td>Iris spatula</td>
			<td>F.S.T</td>
			<td>10092-12</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine (200 mM)</td>
			<td>Gibco</td>
			<td>41010026</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>322412-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>G5767</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting Medium without</td>
			<td>GRIGENE. Ltd, Israel</td>
			<td>K239320A</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraquat</td>
			<td>Sigma-Aldrich</td>
			<td>856177-250MG</td>
			<td>Paraquat dichloride (Methyl viologen)</td>
		</tr>
		<tr>
			<td>PARG- Poly(ADP-Ribose) Glycohydrolase inhibitor</td>
			<td>Tocris Bioscience, United Kingdom</td>
			<td>PDD 00017273</td>
			<td>light sensitive&#160;</td>
		</tr>
		<tr>
			<td>Phosphate buffer</td>
			<td>Biological Indo.</td>
			<td>02-018-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors, skin-tissue</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Six-well plates</td>
			<td>Corning incorporated</td>
			<td>CLS3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Spare Chopping Discs&#160;</td>
			<td>TED PELLA</td>
			<td>Model 10180-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue chopper</td>
			<td>McIlwain</td>
			<td>Model TC752</td>
			<td>10180-220</td>
		</tr>
		<tr>
			<td>Tweezers, pointed</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Urinary cups</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa fluor 488 donkey anti Rabbit IgG</td>
			<td>&#160; 
			Invitrogen</td>
			<td>A21206</td>
			<td>1:500; secondary ab; Secondary dilution buffer;&#160; Incubation for 2 h at room temperature</td>
		</tr>
		<tr>
			<td>Anti glial fibrillary acidic protien</td>
			<td>Milliphore</td>
			<td>MAB3402</td>
			<td>1:1500; primary ab; blocking; Host:mouse</td>
		</tr>
		<tr>
			<td>Anti-calbindin-D-28K</td>
			<td>Swant&#160;</td>
			<td>CB300</td>
			<td>1:2000; primary ab; blocking; host: rabbit&#160;</td>
		</tr>
		<tr>
			<td>Anti-calbindin-D-28K</td>
			<td>Swant</td>
			<td>CB-38a</td>
			<td>1:2000; primary ab; blocking&#160;</td>
		</tr>
		<tr>
			<td>Anti-pan-ADP-ribose reagent</td>
			<td>Milliphore</td>
			<td>MABE1016</td>
			<td>1:800; primary ab; blocking; incubation for 2 h at room temperature</td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidine-2&#39;-phenylindole dihydrochloride)</td>
			<td>Cell signaling</td>
			<td>4084</td>
			<td>1:1000; secondary dilution buffer; incubation for 20 min at room temperature</td>
		</tr>
	</tbody>
</table>

visualizing the dna damage response in purkinje cells using cerebellar organotypic cultures

Cerebellar Purkinje cells (PCs) exhibit a unique interplay of high metabolic rates, specific chromatin architecture, and extensive transcriptional activity, making them particularly vulnerable to DNA damage. This necessitates an efficient DNA damage response (DDR) to prevent cerebellar degeneration, often initiated by PC dysfunction or loss. A notable example is the genome instability syndrome, ataxia-telangiectasia (A-T), marked by progressive PC depletion and cerebellar deterioration. Investigating DDR mechanisms in PCs is vital for elucidating the pathways leading to their degeneration in such disorders. However, the complexity of isolating and cultivating PCs in vitro has long hindered research efforts. Murine cerebellar organotypic (slice) cultures offer a feasible alternative, closely mimicking the in vivo tissue environment. Yet, this model is constrained to DDR indicators amenable to microscopic imaging. We have refined the organotypic culture protocol, demonstrating that fluorescent imaging of protein-bound poly(ADP-ribose) (PAR) chains, a rapid and early DDR indicator, effectively reveals DDR dynamics in PCs within these cultures, in response to genotoxic stress.

Author Spotlight: Deciphering the Role of ATM in Ataxia-Telangiectasia and the Associated Cerebellar Degeneration

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

Our work is focused on understanding a human genetic disorder called ataxia-telangiectasia, or A-T for short. It is caused by loss of the protein kinase ATM. ATM has many functions and A-T is characterized by many symptoms, but the major symptom of A-T is progressive cerebellar degeneration and it is currently unclear which of the many functions of the ATM protein is the one whose loss is specifically responsible for this devastating symptom.We're trying to investigate this central problem in A-T research and for this we are utilizing cerebellar organotypic cultures derived from wild-type and ATM-deficient mice. They allow us to focus on the cell type that is probably the first to be lost in the degenerating cerebellum of A-T patients, Purkinje cells. Like many fields in life sciences research in A-T leveraging a variety of advanced techniques and methods.Those methods include animal models, novel types of tissue cultures, induced polyploid stem cells, cutting-edge imaging technologies, induced omic methods and single cell partials imaging. The source of cerebellar deterioration in A-T is deterioration of Purkinje cells. Those cells are notoriously difficult to isolate and cannot survive in cultures without supporting of other cell types.A unique experimental challenge is obtaining mature, functioning human Purkinje cells using induced polyploid stem cell technology. Since growing isolated Purkinje cells is currently impossible, cerebellar organotypic cultures provide valuable opportunity to study those cells in a culture dish within the natural tissue context. Using protein correlation as a readout for DNA damage response is highly informative, particularly concerning Purkinje cells.A major function of the ATM protein kinase is regulating the DNA damage response, which is critical for maintaining genome integrity and cellular homeostasis in the face of the ongoing DNA damage which occurs in every cell. We need to pinpoint the critical DNA lesion whose repair is defective in ATM-deficient Purkinje cells and we hope to do this using the cerebellar organotypic cultures. This should allow us to understand better the physiological and molecular basis of the most important and devastating symptom of A-T, the cerebellar degeneration.

Figure 1&#160;illustrates the general appearance of the cerebellar organotypic cultures. The top row in Figure 1A shows the cerebellar foliation, which is maintained in culture, while the bottom row shows the PCs stained for Calbindin D-28k (green) and the neuronal nuclei stained for NeuN (red). In Figure 1B, the astrocytes in the PCs (red) are stained for GFAP (green), an intermediate filament type III protein. <strong class="xfig"...

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.

Cerebellar Purkinje cells (PCs) exhibit a unique interplay of high metabolic rates, specific chromatin architecture, and extensive transcriptional ...

visualizing-dna-damage-response-purkinje-cells-using-cerebellar

Research

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JoVE Journal

Biology

225 ビュー.  Tel Aviv University. 私たちの研究は、毛細血管拡張性運動失調症、または略してA-Tと呼ばれるヒトの遺伝性疾患を理解することに焦点を当てています。ATMには多くの機能があり、A-Tには多くの症状がありますが、A-Tの主な症状は進行性の小脳変性であり、ATMタンパク質の多くの機能のうち、どの機能がこの壊滅的な症状に特異的に関与しているのかは現在のところ?...

ビデオ: 著者スポットライト:毛細血管拡張性失調症とそれに伴う小脳変性症におけるATMの役割の解読

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...

https://cloudfront.jove.com/CDNSource/teasers/67167_b.jpg

a simple composite phenotype scoring system for evaluating mouse models of cerebellar ataxia

A Simple Composite Phenotype Scoring System for Evaluating Mouse Models of Cerebellar Ataxia

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebellar ataxia. Measures include hind limb clasping, ledge test, gait and kyphosis. This protocol effectively discriminates between affected and non-affected individuals, and detects the progression of affected individuals over...

modeling neuronal death and degeneration in mouse primary cerebellar granule neurons

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

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...

operational and intervention effects of targeted tuina in lumbar intervertebral disc degeneration model rabbits

Author Spotlight: Investigating the Efficacy and Mechanisms of Tuina Therapy for Intervertebral Disc Degeneration

The finger tactile measurement system quantifies tactile pressure on the finger using a highly sensitive electrosurgical pressure sensor, with software displaying and accurately recording the pressure data and video in real time. Two analysis modules unify the data processing for thumb tracking and conditioning during...

Targeted Tuina Stimulation Protocol in Lumbar Region of IDD Model Rabbits

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examining the role of nasopharyngeal-associated lymphoreticular tissue (nalt) in mouse responses to vaccines

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue NALT in Mouse Responses to Vaccines

Methods to examine contributions of the nasopharyngeal-associated lymphoreticular tissues (NALT) to nasal and systemic immune responses of mice to intranasal vaccines are described.  We demonstrate a surgical procedure to establish a NALT-dependent mouse model and ex vivo cultures of extracted...

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

a mouse model to investigate the role of cancer-associated fibroblasts in tumor growth

A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth

A protocol to co-inject cancer cells and fibroblasts and monitor tumor growth over time is provided. This protocol can be used to understand the molecular basis for the role of fibroblasts as regulators of tumor...

robust mitochondrial isolation from rodent cardiac tissue 

Author Spotlight: Uncovering the Role of Mitochondrial Calcium Phosphate in Heart Failure and Bioenergetics

Bioenergetic and metabolomic studies on mitochondria have revealed their multifaceted role in many diseases, but the isolation methods for these organelles vary. The method detailed here is capable of purifying high-quality mitochondria from multiple tissue sources. Quality is determined by respiratory control ratios and other metrics assessed with high-resolution...

Mitochondrial Isolation from Rodents for High-Quality Bioenergetic Studies

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utilizing murine inducible telomerase alleles in the studies of tissue degeneration/regeneration and cancer

Utilizing Murine Inducible Telomerase Alleles in the Studies of Tissue Degeneration/Regeneration and Cancer

Telomere and telomerase play essential roles in ageing and tumorigenesis. The goal of this protocol is to show how to generate two murine inducible telomerase knock-in alleles and how to utilize them in the studies of tissue degeneration/regeneration and cancer.

introductory analysis and validation of cut&#38;run sequencing data

Author Spotlight: Investigating the Role of Repetitive DNA Misregulation in Cancer Initiation and Immunotherapy Resistance

This protocol guides bioinformatics beginners through an introductory CUT&amp;RUN analysis pipeline that enables users to complete an initial analysis and validation of CUT&amp;RUN sequencing data. Completing the analysis steps described here, combined with downstream peak annotation, will allow users to draw mechanistic insights into chromatin...

Beginner-Friendly Single-Language Pipeline for CUT&RUN Data Analysis

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detection of dna breaks in dividing human cells by neutral comet assay

Author Spotlight: Unveiling the Role of SNF2L in Replication Fork Stability and Genome Duplication

This protocol is designed to investigate the extent of DNA damage occurring during DNA replication. Under neutral conditions, the induction of DNA breaks can be readily assessed within a short time frame. Additionally, the protocol is adaptable to other cell types and various replication stress...

Investigating the Extent of DNA Breaks Occurring During DNA Replication 

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testing the role of multicopy plasmids in the evolution of antibiotic resistance

Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

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visualization of twitching motility and characterization of the role of the pilg in xylella fastidiosa

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

In this study, a nano-microfluidic flow chamber was employed to visualize and functionally characterize the twitching motility of Xylella fastidiosa, a bacterium that causes Pierce&#39;s disease in...

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heat tolerance assays using the drosophila activity monitor system: a guide to an executable application for data analysis

Author Spotlight: Behavioral Thermoregulation and the Role of Automated Analysis in Ectothermic Studies

This report describes a method for measuring adult Drosophila melanogaster time to knockdown using a Drosophila Activity Monitor (DAM2) in response to an air conduction heat stressor within an incubator chamber. The DAM2 measures activity by recording individual fly movements as they cross an infrared beam. Data analysis is facilitated by a novel executable file created by the...

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application of microwave ablation in laparoscopic partial splenectomy

Author Spotlight: Optimizing Hemostasis and Reducing Complications in Laparoscopic Partial Splenectomy &#8211 The Role of Microwave Ablation

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Reducing Bleeding in Laparoscopic Partial Splenectomy with Microwave Ablation

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transgene expression in cultured cells using unpurified recombinant adeno-associated viral vectors

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Recombinant adeno-associated virus (rAAV) is widely used for clinical and preclinical gene delivery. An underappreciated use for rAAVs is the robust transduction of cultured cells without the need for purification. For researchers new to rAAV, we provide a protocol for transgene cassette cloning, crude vector production, and cell culture transduction.

Crude Vector Production and Efficient DNA Delivery in Cell Cultures

lipidunet-machine learning-based method of characterization and quantification of lipid deposits using ipsc-derived retinal pigment epithelium

Author Spotlight: Unraveling the Pathogenesis of Age-Related Macular Degeneration and Discovering Potential Therapies 

Degenerative eye diseases that affect the retinal pigment epithelium layer of the eye have monogenic and polygenic origins. Several disease models and a software application, LipidUNet, have been developed to study mechanisms of disease, as well as potential therapeutic...

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system for focal, closed-system central nervous system injury

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Custom Overpressure Air System for Inducing CNS Injuries in Murine Models

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derivation of thymic lymphoma t-cell lines from atm-/- and p53-/- mice

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

In this video we demonstrate a protocol to establish mouse thymic lymphoma cell lines. By following this protocol, we have successfully established several T-cell lines from Atm-/- and p53-/- mice with thymic...

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Isolation of Cerebellum from Mouse Pups for Obtaining Organotypic Cultures to Study DNA Damage Response

To begin, add one milliliter of basal medium eagle to each well of the six-well plate. And using sterile forceps, place a single cell culture insert into each well. Incubate the plate in a 37 degrees Celsius, 5%carbon dioxide incubator for a minimum of two hours.After setting up the dissection area within a biological hood, wash the surgical tools in 70%ethanol, followed by PBS. Then, position the euthanized animal in the dissection area and make a small incision with scissors away from the cerebellum to expose the brain. Using tweezers with rounded ends, gently peel off the skull and remove the skull in small pieces to avoid damaging the cerebellar tissue.Expose the brain tissue to extract the cerebellum and disconnect the three pairs of peduncles using a fine curved iris spatula to separate the cerebellar cortex from the brain stem. Then, slide the spatula between the cerebellum, the superior colliculus, and the brain stem to retrieve the cerebellum.

Preparation of Cerebellar Organotypic Cultures for Investigating DNA Damage Response in Purkinje Cells

To begin, obtain the cerebellum from a 10-day old mouse pup in cold dissecting buffer. After removing the excess buffer, position the cerebellum on the cutting platform in a vertical orientation. Carry out parasagittal slicing at a slice thickness of 350 micrometers.Gently separate the slices with a fine iris spatula and place them into cold dissection medium. Under the binocular, use two curved iris spatulas to carefully separate the tissue slices from each other. For cultures, use slices derived from the cerebellar vermis situated in the medial corticonuclear zone of the organ.Using a wide spatula, transfer each tissue slice onto a culture insert. Move each insert carrying a tissue slice into a well in the six-well plate containing the pre-warmed medium. During the incubation, aspirate the used medium with a sterile glass pipette.Using a five milliliter serological pipette, add one milliliter of fresh basal medium eagle with the pipette's tip leaning against the well's wall without disturbing the tissue.

Treatment of Cerebellar Organotypic Cultures with DNA Damaging Agents for Fluorescent Imaging of PAR Chains to Assess Genotoxic Stress Response

To begin, culture the chopped cerebellar tissue retrieved from 10 day-old mouse pup in basal medium eagle. Add DNA damaging chemicals directly to the culture medium at appropriate final concentrations. After the exposure period, replace the medium with a freshly prepared mixed medium containing the DNA damaging chemicals and the poly ADP ribose glycohydrolase inhibitor, and incubate for 30 minutes.After 30 minutes, wash the slices with 0.1 molar phosphate buffer at room temperature and immediately fix them with a pre-cooled acetone and methanol mixture. Incubate the plate for 20 minutes at minus 20 degrees Celsius. Then remove the fixation solution and wash it with 0.4 molar phosphate buffer.Add 100 microliters of 0.1 molar phosphate buffer to each well of a 48 well plate. Then use a scalpel and a cutting board to remove the margins of the insert, cutting around the tissue slice. Place the slice in a well of the 48 well plate.After aspirating the buffer, incubate the insert with 100 microliters of 0.1 molar phosphate buffer containing 1%Triton X100. Aspirate the solution and serially incubate the insert in each well on a shaker with appropriate amounts of blocking solution followed by the primary in the secondary antibodies. Add 20 microliters of the final DAPI solution to each well 20 minutes before the end of the two hour incubation with the secondary antibody and continue shaking for 20 minutes.For mounting, place the tissue on a microscope glass slide with the tissue facing up. Add the mounting medium and cover with a cover glass. Place the slides on a flat surface and let them dry overnight in the dark at four degrees Celsius.The cerebellar foliation was maintained in the culture. Purkinje cells were stained for Calbindin D-28K and neural nuclei stained for NeuN. The astrocytes in the Purkinje cells were stained for GFAP.PAR staining increased in treated Purkinje cells after exposure to potassium bromate and paraquat indicating DNA damage.