Sriharsa Pradhan, Shannon Morey Kinney, Hang Gyeong Chin, Jurate Bitinaite, Yu Zheng, Pierre Olivier Esteve, Romualdas Vaisvila, Steven E. Jacobsen s Labor, UCLA. Diese Arbeit wurde teilweise durch NIH 1R44GM095209-01 unterstützt.

1. DNA Glucosylierung and Control Reaktionen <ol><li> In einem 1,5 ml Reaktionsgefäß auf Eis mischen 5-10 Mikrogramm der genomischen DNA (für eine endgültige Konzentration von 30 Mikrogramm / Milliliter), 12,4 Mikroliter UDP-Glucose (für eine endgültige Konzentration von 80 Mikromol), 31 Mikroliter NEBuffer 4 und bis zu 310 Mikroliter Nuklease-freies Wasser zu bringen das Gesamtvolumen auf 310 Mikroliter. </li><li> Split dieses Reaktionsgemisch in zwei Röhren von 155 Mikroliter jeder. </li><li> Dann fügen Sie 30 Einheiten oder 3 Mikroliter, der T4-β-glucosyltransferase zu einer Röhre. Gut mischen durch vorsichtiges Auf-und Abpipettieren. Die zweite Röhre wird der Kontroll-Reaktion, so fügen Sie 3 Mikroliter Wasser anstelle von T4-BGT. </li><li> Inkubieren beiden Röhren bei 37 Grad Celsius für 12 bis 18 Stunden, während welcher Zeit der T4-BGT wird Glucose an die Hydroxylgruppe von 5-Hydroxymethylcytosin Gruppen in der Probe hinzufügen. </li></ol> 2. Restriktionsenzymverdau <ol><li> Label drei 0,2 ml PCR-Streifen Rohre Nummern 1 bis 3. Aliquot 50 ul des Reaktionsgemisches in jedem. </li><li> Label drei 0,2 ml PCR-Streifen Rohre Nummern 4 bis 6 aus. Aliquot 50 ul der Kontrolle Mischung in jeder. </li><li> Add 100 Einheiten, oder 1 Mikroliter, der MspI in Röhrchen 1 und 4. Gut mischen durch vorsichtiges Auf-und Abpipettieren. </li><li> Fügen Sie 50 Einheiten, oder 1 Mikroliter, der HpaII in Rohren 2 und 5. Gut mischen durch vorsichtiges Auf-und Abpipettieren. </li><li> Fügen Sie nicht etwas zu Röhrchen 3 und 6, da sie steuert werden. </li><li> Inkubieren Sie alle 6 Röhren bei 37 Grad Celsius für mindestens 4 Stunden. </li><li> Add 1 Mikroliter Proteinase K in jedes Röhrchen und inkubieren Sie bei 40 Grad Celsius für 30 Minuten. </li><li> Inaktivierung der Proteinase K durch Inkubation bei 95 Grad Celsius für 10 Minuten. </li></ol> 3. Analysieren DNA durch PCR / qPCR Dieses Protokoll verwendet NEB ist LongAmp Taq für Endpunkt PCR, die gezeigt worden ist, eine gute Leistung. Andere PCR-Protokolle ersetzt werden können. <ol><li> Fügen Sie die folgenden Komponenten, um eine 0,2 Milliliter PCR-Reaktionsgefäß auf Eis: 10 Mikroliter 5X LongAmp Taq-Reaktionspuffer, 1,5 Mikroliter 10 mM dNTPs, 1 Mikroliter von 10 Mikromol Vorwärtsprimer, 1 Mikroliter von 10 Mikromol Reverse-Primer, 3 Mikroliter Vorlage DNA aus den vorherigen Schritten, 1 Mikroliter LongAmp Taq DNA Polymerase und Nuklease-freies Wasser bis zu 50 Mikroliter. Diese Mengen können je nach PCR-Protokoll zu ändern. </li><li> Vorsichtig mischen die Reaktion. Wenn nötig, sammeln Sie alle Flüssigkeit auf den Boden der Röhre durch eine schnelle Runde. </li><li> Overlay der Probe mit Mineralöl bei Verwendung eines Thermocycler ohne Heizdeckel. </li><li> Übertragen Sie die PCR-Röhrchen von Eis zu einem Thermocycler mit dem Block vorgeheizt auf 94 Grad Celsius und starten Sie den Radsport-Programm. Für eine Routine 3-Schritt-PCR, sollte es eine initiale Denaturierung bei 94 Grad Celsius für 30 Sekunden von 30 Zyklen von 94 Grad Celsius Denaturierung für 15 Sekunden, 55 bis 65 Grad Celsius Annealing für 30 Sekunden und 65 Grad Celsius Erweiterung folgen für 20 Sekunden oder 50 Sekunden pro kb. Dies sollte durch eine letzte Verlängerung bei 65 Grad Celsius für 5 Minuten eingehalten werden. Real-time PCR kann auch durchgeführt werden, um Mengen von 5-Methyl-und 5-Hydroxymethylcytosin quantifizieren sein. Bitte beachten Sie die Produkt-Handbuch, auf neb.com gefunden für spezifische Informationen. </li></ol> 4. Repräsentative Ergebnisse: <img src="/files/ftp_upload/2661/2661fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Vergleich von 5-Hydroxymethylcytosin Mengen an Locus 12 in verschiedenen Maus Balb / C Gewebeproben. (A), End-Point-PCR. (B), Real time PCR. DNA aus vier Mausgeweben analysiert. Zum Zwecke der Vergleichbarkeit wurden real time PCR Daten uncut DNA normalisiert. Eine Standardkurve wurde verwendet, um die Nummer des Exemplars zu bestimmen. Die Proben konnten, indem man die Kopienzahl der Proben No 1-6 durch die Kopienzahl der Kontrolle, ist unverdaut (Nr. 5) normalisiert werden. Boxed Gelspur zeigt Variante in 5-HMC präsentieren.

<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>

Beide Ted und Romas, sind Autoren dieses Artikels, Mitarbeiter der NEB; Romas ist der Hauptentwickler des Kits, mit Ted wird der Direktor des Applications &amp; Development Department, dass die Entwicklung dieses Kit überwachte.

Es gibt einige kritische Dinge zu beachten bei der Einrichtung dieses Experiment. Zunächst ist es wichtig, dass die Glucosylierung der genomischen DNA vollständig verläuft. Die Sequenz-Spezifität der T4-BGT ist nicht bekannt, und es scheint keine andere Wahl für das Substrat Folge haben. Daher ist in einigen Fällen kann längere Inkubationszeiten notwendig sein. Zweitens MspI und HpaII Verdauung muss, um Hintergrund-Signal zu vermeiden abzuschließen. Für diese Restriktionsenzyme, empfehlen wir eine Inkubationszeit von 4 Stunden, aber mehr Inkubationen können durchgeführt werden, wenn unvollständige Spaltung beobachtet werden. Drittens kann Input DNA-Menge angepasst je nach Verfügbarkeit, seit 20 ngs der DNA für die Endpunkt-PCR verwendet werden kann. Schließlich empfehlen wir die Nutzung der mitgelieferten Kontroll-DNA, die parallel mit genomischer DNA für 5-mC und 5-HMC Quantifizierung kann ausgeführt werden.

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		<th>Material Name</th>
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		<th>Catalogue Number</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

DNA Hydroxymethylierung ist eine lange bekannte Modifizierung von DNA, aber seit kurzem ein Schwerpunkt in der epigenetischen Forschung. Mammalian DNA wird enzymatisch an der 5 th Kohlenstoff-Position von Cytosin (C)-Reste bis 5-mC geändert, überwiegend im Zusammenhang mit der CpG-Dinukleotide. 5-mC zugänglich ist enzymatische Oxidation zu 5-HMC von der Tet-Familie von Enzymen, die vermutlich in Entwicklung und Krankheit beteiligt sind. Derzeit ist die biologische Rolle von 5-HMC nicht vollständig verstanden, sondern ist auf großes Interesse, da ihr Potential als Biomarker. Dies ist auf mehrere bahnbrechende Studien zu identifizieren 5-Hydroxymethylcytosin in embryonalen Maus-Stammzellen (ES) und neuronalen Zellen. Forschung Techniken, einschließlich Bisulfit-Sequenzierung Methoden sind nicht in der Lage, problemlos zwischen 5-mC-und 5-hmc unterscheiden. Ein paar Protokolle existieren, die Gesamtbeträge von 5-Hydroxymethylcytosin in das Genom zu messen, einschließlich Flüssigchromatographie mit Massenspektrometrie-Analyse oder Dünnschichtchromatographie einzelner Nucleoside aus genomischer DNA verdaut gekoppelt. Antikörper, die auf 5-Hydroxymethylcytosin gibt es auch, die für Dot-Blot-Analyse, Immunfluoreszenz oder Fällung von hydroxymethylierten DNA verwendet werden, können aber diese Antikörper nicht haben Einzel-Basis resolution.In Darüber hinaus hängt die Auflösung auf die Größe des immunpräzipitiert DNA und für Microarray-Experimenten, hängt davon ab, Sonde Design. Da nicht bekannt ist, wo genau 5-Hydroxymethylcytosin in das Genom oder seine Rolle in der epigenetischen Regulation existiert, sind neue Techniken erforderlich, die spezifischen Locus Hydroxymethylierung identifizieren. Die EpiMark 5-HMC und 5-mC Analysis Kit bietet eine Lösung für die Unterscheidung zwischen diesen beiden Änderungen an bestimmten Orten. Die EpiMark 5-HMC und 5-mC Analysis Kit ist eine einfache und robuste Methode zur Identifizierung und Quantifizierung von 5-Methyl-und 5-Hydroxymethylcytosin innerhalb eines bestimmten DNA-Locus. Diese enzymatische Ansatz nutzt die Differential-Methylierung Empfindlichkeit des Isoschizomere MspI und HpaII in einer einfachen 3-Schritt-Protokoll. Genomic DNA von Interesse ist mit T4-BGT behandelt, indem eine Glukose moeity bis 5-Hydroxymethylcytosin. Diese Reaktion ist Sequenz-unabhängige, also alle 5-HMC glucosylierten werden; unmodifizierte oder 5-mC mit DNA wird dadurch nicht beeinflusst. Diese Glucosylierung wird dann durch Restriktionsendonukleaseverdau gefolgt. MspI und HpaII erkennen die gleiche Sequenz (CCGG), sind aber empfindlich auf unterschiedliche Methylierung Staaten. HpaII spaltet nur einen komplett unveränderten Ort: jede Änderung (5-mC, 5-HMC-oder 5-ghmC) an beiden Cytosin-Blöcke Spaltung. MspI erkennt und schneidet 5-mC-und 5-HMC, aber nicht 5-ghmC. Der dritte Teil des Protokolls ist eine Abfrage der Locus durch PCR. So wenig wie 20 ng der DNA-Eingang genutzt werden kann. Verstärkung der experimentellen (glucosylierten und verdaut) und Kontrolle (mock glucosylierten und verdaut) Ziel-DNA mit Primer flankieren CCGG Ort von Interesse (100-200 bp) durchgeführt wird. Wenn der CpG site enthält 5-Hydroxymethylcytosin, ist eine Band nach Glucosylierung und Verdauung erkannt, aber nicht in den nicht-glucosylierten Kontroll-Reaktion. Real-time PCR gibt eine Annäherung, wie viel Hydroxymethylcytosin ist in diesem besonderen Ort. In diesem Experiment werden wir die 5-Hydroxymethylcytosin Betrag in eine Maus Babl / C Gehirn Probe zu analysieren, indem Endpunkt PCR.

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

Die EpiMark 5-HMC und 5-mC Analysis Kit kann verwendet werden, um zu analysieren und zu quantifizieren 5-Methylcytosin und 5-Hydroxymethylcytosin innerhalb einer spe cific locus werden. Das Kit unterscheidet 5-mC von 5-HMC von der Zugabe von Glucose an die Hydroxylgruppe von 5-HMC über eine enzymatische Reaktion unter Verwendung von β-glucosyltransferase (T4-BGT). Als 5-HMC im Rahmen der CCGG auftritt, wandelt diese Modifikation eines spaltbaren MspI Ort zu einer nicht-spaltbare Website.

Hohe Empfindlichkeit 5-Hydroxymethylcytosin Erkennung in Balb / C Gehirngewebe

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

high-sensitivity-5-hydroxymethylcytosine-detection-balbc-brain

Research

JoVE Journal

Neuroscience

14.9K Aufrufe.  New England Biolabs. Die EpiMark 5-HMC und 5-mC Analysis Kit kann verwendet werden, um zu analysieren und zu quantifizieren 5-Methylcytosin und 5-Hydroxymethylcytosin innerhalb einer spe cific locus werden. Das Kit unterscheidet 5-mC von 5-HMC von der Zugabe von Glucose an die Hydroxylgruppe von 5-HMC über eine enzymatische Reaktion unter Verwendung von β-glucosyltransferase (T4-BGT). Als 5-HMC im Rahmen der CCGG auftritt, wandelt diese Modifikation eines spaltbaren MspI Ort zu einer nicht-...

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

A Simple Way to Measure Ethanol Sensitivity in Flies

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain is correlated with that strain s ethanol preference, and therefore sedation is a highly relevant measure to study the genetics of alcohol responses and drinking. We demonstrate a simple way to expose flies to ethanol and measure its intoxicating effects. The assay we describe can determine acute sensitivity, as well as ethanol tolerance induced by repeat exposure. It does not require a technically involved setup, and can therefore be applied in any laboratory with basic fly culture tools.

A simple assay to measure the sedating effects of ethanol on Drosophila flies, based on the loss of righting reflex, is described.

Ein einfacher Weg zu Ethanol Empfindlichkeit in Flies Measure

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain ...

Forschung

Neurowissenschaften

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.

Detektion von Protein Palmitoylierung in kultivierten Hippocampus-Neuronen durch Immunopräzipitation und 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 ...

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).

Metabolomische Analyse von Rattenhirn Hohe Auflösung Kernresonanzspektroskopie von Gewebeextrakte

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.

Eine einfache und kostengünstige Methode zur Bestimmung der Überempfindlichkeit auf Kälte und Anpassung an der Maus

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.

Nachweis von Axonally lokalisierten mRNAs in Gehirnschnitten mittels hochauflösender<em&gt; In-situ-</em&gt; Hybridization

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 von DAN Matrix für die Erkennung und Visualisierung von Gangliosiden in Rattenhirngewebe für die MALDI-Imaging-Massenspektrometrie

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.

Hochdurchsatz-Analyse des Bewegungsapparates Verhaltens in der Drosophila -Insel-Assay

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.

Hochauflösende 3D-Bildgebung der Tollwut-Virusinfektion im lösungsmittelfreien Hirngewebe

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.

Einsatz von Infrarot-Fluoreszenz und hochauflösendem Scannen zur Messung des Protein-Expression im Rodentrains

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 ...