Sriharsa Pradhan, Shannon Morey Kinney, Hang Gyeong Chin, Jurate Bitinaite, Yu Zheng, Pierre Olivier Estève, Romualdas Vaisvila, laboratoire Steven E. Jacobsen s, UCLA. Ce travail a été partiellement financé par le NIH 1R44GM095209-01.

1. Glucosylation ADN et contrôler les réactions <ol><li> Dans un tube de réaction 1,5 millilitre sur la glace, mélanger microgrammes 5-10 de l&#39;ADN génomique (pour une concentration finale de 30 microgrammes / millilitre), 12,4 microlitres de l&#39;UDP-glucose (pour une concentration finale de 80 micromolaire), 31 microlitres de NEBuffer 4 et jusqu&#39;à 310 microlitres eau sans nucléase pour porter le volume total à 310 microlitres. </li><li> Fractionner ce mélange réactionnel dans deux tubes de 155 microlitres chacun. </li><li> Puis ajouter 30 unités, soit 3 microlitres, de T4-β-glucosyltransférase à un tube. Bien mélanger en douceur aspiration et refoulement. Le deuxième tube est la réaction de contrôle, donc ajoutez 3 microlitres d&#39;eau, au lieu de T4-BGT. </li><li> Incuber les deux tubes à 37 degrés Celsius pendant 12 à 18 heures, période pendant laquelle le T4-BGT va ajouter de glucose pour le groupe hydroxyle de 5-hydroxyméthylcytosine groupes de l&#39;échantillon. </li></ol> 2. Digestion enzymatique de restriction <ol><li> Étiquette trois 0,2 millilitre PCR bande numéros 1 à 3 tubes. Aliquote de 50 ul du mélange réactionnel dans chaque. </li><li> Étiquette trois 0,2 millilitre PCR bande numéros tubes 4 à 6. Aliquote de 50 ul du mélange de contrôle dans chaque. </li><li> Ajouter 100 unités, soit 1 microlitre, des Mspl dans des tubes 1 et 4. Bien mélanger en douceur aspiration et refoulement. </li><li> Ajouter 50 unités, soit 1 microlitre de Hpall dans des tubes de 2 et 5. Bien mélanger en douceur aspiration et refoulement. </li><li> Ne pas ajouter quoi que ce soit à des tubes 3 et 6, car ils sont les témoins. </li><li> Incuber tous les tubes de 6 à 37 degrés Celsius pendant au moins 4 heures. </li><li> Ajouter 1 microlitre de la protéinase K dans chaque tube et incuber à 40 degrés Celsius pendant 30 minutes. </li><li> Inactiver la protéinase K par une incubation à 95 degrés Celsius pendant 10 minutes. </li></ol> 3. Analyser l&#39;ADN par PCR / qPCR Ce protocole utilise ONÉ LongAmp Taq pour point final de PCR qui a été montré à bien performer. D&#39;autres protocoles de PCR peut être substitué. <ol><li> Ajouter les éléments suivants dans un tube de 0,2 ml PCR sur glace: 10 microlitres de tampon de réaction 5X LongAmp Taq, 1,5 microlitres de dNTP 10 mM, 1 microlitre de 10 amorce sens micromolaires, 1 microlitre de 10 amorce reverse micromolaires, 3 microlitres de template l&#39;ADN à partir des étapes précédentes, 1 microlitre de la polymérase Taq ADN LongAmp, et une eau sans nucléase jusqu&#39;à 50 microlitres. Ces volumes peuvent changer selon le protocole de PCR utilisées. </li><li> Mélanger délicatement la réaction. Si nécessaire, recueillir tout le liquide au fond du tube par un tour rapide. </li><li> Superposition de l&#39;échantillon avec de l&#39;huile minérale si vous utilisez un thermocycleur sans couvercle chauffant. </li><li> Transférer les tubes de PCR de la glace à un thermocycleur avec le bloc préchauffé à 94 degrés Celsius et démarrer le programme de cyclisme. Pour une routine 3-étape de PCR, il devrait y avoir une étape de dénaturation initiale à 94 degrés Celsius pendant 30 secondes, suivi par 30 cycles de 94 degré Celsius la dénaturation pendant 15 secondes, 55 à 65 degrés Celsius recuit pendant 30 secondes, et 65 degré Celsius d&#39;extension pendant 20 secondes ou 50 secondes par ko. Cela devrait être suivi par une extension finale à 65 degrés Celsius pendant 5 minutes. PCR en temps réel peut également être réalisée pour quantifier des quantités de 5-méthylcytosine et 5-hydroxyméthylcytosine. S&#39;il vous plaît se référer à la notice du produit, trouvé sur neb.com pour des informations spécifiques. </li></ol> 4. Les résultats représentatifs: <img src="/files/ftp_upload/2661/2661fig1.jpg" alt="Figure 1" /> Figure 1. Comparaison de la 5-hydroxyméthylcytosine sommes au locus 12 dans des échantillons de tissus de souris différents Balb / C. (A), point final de PCR. (B), PCR temps réel. L&#39;ADN à partir de quatre tissus de la souris a été analysée. Pour fins de comparaison, PCR en temps réel les données ont été normalisées à l&#39;ADN non coupé. Une courbe standard a été utilisé pour déterminer le nombre de copies. Les échantillons peuvent être normalisées en divisant le nombre de copies d&#39;échantillons Aucun 1-6 par le nombre de copies du contrôle qui est non digérées (no 5). Voies de gel Boxed montre la variation en 5-HMC présents.

<ol>
	<li>Sadri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nucleic Acids Res. 24, 4987-4989 (1996).</li><li>Mathur, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Gene. 108, 1-6 (1991).</li><li>Butkus, V., Petrauskiene, L., Maneliene, Z., Klimasaukas, S., Laucys, V., Janulaitis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nucleic Acids Res. 15, 7091-7102 (1987).</li><li>Kriaucionis, S., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Science. 324, 929-930 (2009).</li><li>Tahiliani, M., Koh, K. P., Shen, Y., Pastor, W. A., Bandukwala, H., Brudno, Y., Agarwal, S., Lyer, L. M., Liu, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Science. 324, 930-935 (2009).</li><li>Huang, Y., Pastor, W. A., Shen, Y., Tahiliani, M., Liu, D. R., Rao, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> PLoS One. 5 (1), e8888-e8888 (2010).</li></ol>

Ted et Romas, les auteurs de cet article, sont des employés de l&#39;ONÉ; Romas est le développeur principal de la trousse, avec Ted étant le directeur de la Direction du Développement Applications &amp; qui a supervisé le développement de ce kit.

Il ya plusieurs choses essentielles à considérer lors de la mise en place de cette expérience. Premièrement, il est important que le glucosylation d&#39;ADN génomique provenant d&#39;achèvement. La spécificité de séquence de T4-BGT n&#39;est pas connue, et il ne semble pas avoir le choix pour la séquence de substrat. Par conséquent, dans certains cas, les temps d&#39;incubation plus longue peut être nécessaire. Seconde digestion, Mspl et Hpall doit être complète afin d&#39;éviter un signal de fond. Pour ces enzymes de restriction, nous recommandons un temps d&#39;incubation de 4 heures, mais plus d&#39;incubations peuvent être réalisées lors clivage incomplet est observée. Troisièmement, le montant ADN d&#39;entrée peut être ajustée en fonction de la disponibilité, depuis le 20 END de l&#39;ADN peuvent être utilisés pour point final de PCR. Enfin, nous recommandons l&#39;utilisation de l&#39;ADN de contrôle fourni, qui peut être exécuté en parallèle avec l&#39;ADN génomique pour la 5-MC et 5-HMC quantification.

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>EpiMark&trade; 5-hmC and 5-mC Analysis Kit</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>E3317</td>
<td>&nbsp;</td>
<tr>
<td>Locus specific primers, flanking a CCGG site of interest</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Custom per experiment; provided by user</td>
</tr>
<tr>
<td>LongAmp&reg; Taq DNA Polymerase</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>M0323</td>
<td>&nbsp;</td>
<tr>
<td>dNTPs</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>N0447</td>
<td>&nbsp;</td>
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<td>molecular biology grade water</td>
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high sensitivity 5-hydroxymethylcytosine detection in balb/c brain tissue

Hydroxyméthylation ADN est une modification de l&#39;ADN connue depuis longtemps, mais est récemment devenu un foyer dans la recherche en épigénétique. L&#39;ADN des mammifères est enzymatiquement modifiés à la position du carbone 5 ème de la cytosine (C) aux résidus 5-MC, principalement dans le contexte des dinucléotides CpG. 5-MC se prête à l&#39;oxydation enzymatique de 5-HMC par la famille du Têt des enzymes, qui sont soupçonnés d&#39;être impliqués dans le développement et la maladie. Actuellement, le rôle biologique de 5-HMC n&#39;est pas entièrement comprise, mais suscite beaucoup d&#39;intérêt en raison de son potentiel en tant que biomarqueur. Cela est dû à plusieurs études novatrices d&#39;identification 5-hydroxyméthylcytosine chez la souris souches embryonnaires (ES) et les cellules neuronales. Les techniques de recherche, y compris les méthodes de bisulfite de séquençage, sont incapables de distinguer facilement entre 5-MC et 5-HMC. Quelques protocoles existent qui peuvent mesurer des montants globaux de la 5-hydroxyméthylcytosine dans le génome, dont la chromatographie liquide couplée à la spectrométrie de masse ou chromatographie sur couche mince de nucléosides seule digéré de l&#39;ADN génomique. Les anticorps qui cible 5-hydroxyméthylcytosine existent également, qui peut être utilisé pour l&#39;analyse dot blot, immunofluorescence, ou à la précipitation d&#39;ADN hydroxyméthylés, mais ces anticorps n&#39;ont pas seule plus resolution.In de base, la résolution dépend de la taille de l&#39;ADN et pour immunoprécipité expériences biopuces, dépend de la conception de la sonde. Comme il ne sait pas exactement où 5-hydroxyméthylcytosine existe dans le génome ou de son rôle dans la régulation épigénétique, de nouvelles techniques sont nécessaires qui peuvent identifier hydroxyméthylation locus spécifique. Le EpiMark 5 HMC et 5-MC Analyse kit offre une solution de distinction entre ces deux modifications à des loci particuliers. Le EpiMark 5-HMC et analyse 5-MC Kit est une méthode simple et robuste pour l&#39;identification et la quantification de la 5-méthylcytosine et 5-hydroxyméthylcytosine dans un locus d&#39;ADN spécifiques. Cette approche enzymatique utilise la sensibilité méthylation différentielle de l&#39;Mspl isoschizomères et Hpall dans un simple en 3 étapes du protocole. L&#39;ADN génomique d&#39;intérêt est traitée avec T4-TBG, l&#39;ajout d&#39;un moeity glucose à 5-hydroxyméthylcytosine. Cette réaction est indépendante de la séquence, donc tous les cinq-HMC sera glucosylés; non modifiés ou 5-MC ADN contenant ne sera pas affectée. Cette glucosylation est alors suivie d&#39;une digestion par endonucléase de restriction. Mspl et Hpall reconnaissent la même séquence (CCBG), mais sont sensibles à la méthylation états différents. Hpall clive seulement un site totalement non modifiée: toute modification (5-mC, 5-HMC ou 5-ghmC) soit à un clivage des blocs de cytosine. Mspl reconnaît et clive 5-MC et 5-HMC, mais pas 5 ghmC. La troisième partie de ce protocole est l&#39;interrogation du locus par PCR. Aussi peu que 20 ng d&#39;ADN d&#39;entrée peuvent être utilisés. Amplification de l&#39;expérimental (glucosylés et digéré) et de contrôle (maquette glucosylés et digéré) avec des amorces d&#39;ADN cible flanquant un site CCBG d&#39;intérêt (100-200 pb) est effectuée. Si le site contient des CpG 5-hydroxyméthylcytosine, une bande est détecté après glucosylation et la digestion, mais pas dans la réaction de contrôle non-glycosylées. PCR en temps réel donne une approximation de combien hydroxyméthylcytosine est dans ce site particulier. Dans cette expérience, nous allons analyser la quantité de 5 hydroxyméthylcytosine dans un échantillon de cerveau de souris Babl / C par PCR point final.

Watch this Scientific Journal Video about High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue at JoVE.com

High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue

Le EpiMark 5-HMC et le kit d&#39;analyse 5-MC peut être utilisé pour analyser et quantifier 5-méthylcytosine et 5-hydroxyméthylcytosine au sein d&#39;un locus de SPE spécifiques. Le kit distingue 5-MC à partir de 5-HMC par l&#39;ajout de glucose pour le groupe hydroxyle de 5-HMC via une réaction enzymatique utilisant β-glucosyltransférase (T4-TBG). Lorsque 5-HMC survient dans le contexte de la CCBG, cette modification convertit un site clivable Mspl à un site non clivable.

Haute Sensibilité 5-hydroxyméthylcytosine détection dans le tissu cérébral des souris Balb / C

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research.   Mammalian DNA is enzymatically ...

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Neuroscience

14.9K vues.  New England Biolabs. Le EpiMark 5-HMC et le kit d'analyse 5-MC peut être utilisé pour analyser et quantifier 5-méthylcytosine et 5-hydroxyméthylcytosine au sein d'un locus de SPE spécifiques. Le kit distingue 5-MC à partir de 5-HMC par l'ajout de glucose pour le groupe hydroxyle de 5-HMC via une réaction enzymatique utilisant β-glucosyltransférase (T4-TBG). Lorsque 5-HMC survient dans le contexte de la CCBG, cette modification convertit un site clivable Mspl à un site no...

Vidéo: High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

Détection de la palmitoylation des protéines dans les neurones de l&#39;hippocampe en culture par immunoprécipitation et Acyl-biotine Exchange (ABE)

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

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Neurosciences

Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics and proteomics have been complemented by comprehensive quantitative analysis of the metabolite pool present in biological systems. This strategy, termed metabolomics, strives to provide a global characterization of the small-molecule complement involved in metabolism. While the genome and the proteome define the tasks cells can perform, the metabolome is part of the actual phenotype. Among the methods currently used in metabolomics, spectroscopic techniques are of special interest because they allow one to simultaneously analyze a large number of metabolites without prior selection for specific biochemical pathways, thus enabling a broad unbiased approach. Here, an optimized experimental protocol for metabolomic analysis by high-resolution NMR spectroscopy is presented, which is the method of choice for efficient quantification of tissue metabolites. Important strengths of this method are (i) the use of crude extracts, without the need to purify the sample and/or separate metabolites; (ii) the intrinsically quantitative nature of NMR, permitting quantitation of all metabolites represented by an NMR spectrum with one reference compound only; and (iii) the nondestructive nature of NMR enabling repeated use of the same sample for multiple measurements. The dynamic range of metabolite concentrations that can be covered is considerable due to the linear response of NMR signals, although metabolites occurring at extremely low concentrations may be difficult to detect. For the least abundant compounds, the highly sensitive mass spectrometry method may be advantageous although this technique requires more intricate sample preparation and quantification procedures than NMR spectroscopy. We present here an NMR protocol adjusted to rat brain analysis; however, the same protocol can be applied to other tissues with minor modifications.

The neurochemistry of mammalian brain is changed in many neurological and systemic diseases. Characteristic profiles of cerebral metabolites can be efficiently obtained based on crude extracts of brain tissue. To this end, high-resolution NMR spectroscopy is employed, enabling detailed quantitative analysis of metabolite concentrations (metabolomics).

Analyse métabolomique de cerveau de rat par la Résolution nucléaire Haute spectroscopie par résonance magnétique d&#39;extraits de tissus

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Une méthode simple et peu coûteuse pour déterminer Sensibilité au froid et adaptation chez la souris

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

Détection de axonaux localisée ARNm dans les sections de cerveau en utilisant haute résolution<em&gt; In Situ</em&gt; Hybridation

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass Spectrometry (IMS) experiments. Determining the appropriate protocol to follow throughout the sample preparation process can be difficult as each step must be optimized to comply with the unique characteristics of the analytes of interest. This process involves not only finding a compatible matrix that can desorb and ionize the molecules of interest efficiently, but also selecting the appropriate matrix deposition technique. For example, a wet matrix deposition technique, which entails dissolving a matrix in solvent, is superior for desorption of most proteins and peptides, whereas dry matrix deposition techniques are particularly effective for ionization of lipids. Sublimation has been reported as a highly efficient method of dry matrix deposition for the detection of lipids in tissue by MALDI IMS due to the homogeneity of matrix crystal deposition and minimal analyte delocalization as compared to many wet deposition methods 1,2. Broadly, it involves placing a sample and powdered matrix in a vacuum-sealed chamber with the samples pressed against a cold surface. The apparatus is then lowered into a heated bath (sand or oil), resulting in sublimation of the powdered matrix onto the cooled tissue sample surface. Here we describe a sublimation protocol using 1,5-diaminonaphthalene (DAN) matrix for the detection and visualization of gangliosides in the rat brain using MALDI IMS.

A protocol for the sublimation of DAN matrix onto rat brain tissue for the detection of gangliosides using MALDI Imaging Mass Spectrometry is presented.

Sublimation de DAN Matrice pour la détection et la visualisation des gangliosides dans Rat Tissue Brain MALDI Imaging Mass Spectrometry

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

Analyse de haut-débit du comportement locomoteur dans le dosage de l’île de drosophile

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample&#8217;s refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

Imagerie 3D haute résolution de l'infection par le virus de la rage dans un tissu cérébral éclairci par solvant

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

Utilisation de la fluorescence proche infrarouge et de la numérisation haute résolution pour mesurer l’expression des protéines dans le cerveau des rongeurs

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

La détection de 5-Hydroxymethylcytosine dans les cellules souches neurales et le cerveau des souris

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...