Sriharsa Pradhan, Shannon Morey Kinney, Hang Chin Gyeong, Jurate Bitinaite, Yu Zheng, Pierre Olivier Esteve, Romualdas Vaisvila, laboratorio di Steven E. Jacobsen s, UCLA. Questo lavoro è stato parzialmente supportato dal NIH 1R44GM095209-01.

1. DNA Glucosylation e controllare le reazioni <ol><li> In un tubo di reazione 1,5 millilitri su ghiaccio, mescolare 5-10 microgrammi di DNA genomico (per una concentrazione finale di 30 microgrammi / millilitro), il 12,4 microlitri di UDP-glucosio (per una concentrazione finale di 80 micromolari), 31 microlitri di NEBuffer 4 e fino a 310 microlitri di nucleasi senza acqua per portare il volume totale a 310 microlitri. </li><li> Split questa miscela di reazione in due tubi di 155 microlitri ciascuno. </li><li> Quindi aggiungere 30 unità, o 3 microlitri, di T4-β-glucosiltransferasi a un tubo. Mescolare bene pipettando gentilmente su e giù. Il secondo tubo è la reazione di controllo, in modo da aggiungere 3 microlitri di acqua, invece di T4-BGT. </li><li> Incubare entrambi i tubi a 37 gradi centigradi per 12 a 18 ore, durante il quale il T4-BGT aggiungerà glucosio per il gruppo ossidrile di 5-hydroxymethylcytosine gruppi del campione. </li></ol> 2. Digestione degli enzimi di restrizione <ol><li> Etichetta tre 0,2 millilitri PCR-strip numeri tubi da 1 a 3. Aliquota 50 ul della miscela di reazione in ogni. </li><li> Etichetta tre 0,2 millilitri PCR-strip numeri tubi da 4 a 6. Aliquota 50 ul della miscela di controllo in ciascuno. </li><li> Aggiungere 100 unità, o 1 microlitro, di MspI in tubi 1 e 4. Mescolare bene pipettando gentilmente su e giù. </li><li> Aggiungere 50 unità, o 1 microlitro, di HpaII in tubi 2 e 5. Mescolare bene pipettando gentilmente su e giù. </li><li> Non aggiunge nulla ai tubi 3 e 6, in quanto sono controlli. </li><li> Incubare tutte le 6 tubi a 37 ° C per almeno 4 ore. </li><li> Aggiungere 1 microlitro di proteinasi K in ciascuna provetta e incubare a 40 ° C per 30 minuti. </li><li> Inattivare il proteinasi K incubando a 95 gradi centigradi per 10 minuti. </li></ol> 3. Analizzare il DNA mediante PCR / qPCR Questo protocollo utilizza NEB è LongAmp Taq per end-point PCR che ha dimostrato di funzionare bene. Altri protocolli di PCR può essere sostituito. <ol><li> Aggiungere i seguenti componenti di una reazione di 0,2 tubo millilitro PCR in ghiaccio: 10 microlitri di tampone di reazione 5X LongAmp Taq, 1,5 microlitri di 10 dNTPs millimolare, 1 microlitro di 10 Primer micromolari Forward, 1 microlitro di 10 Primer Reverse micromolari, 3 microlitri di template DNA dai passaggi precedenti, 1 microlitro di Taq DNA polimerasi LongAmp e acqua priva di nucleasi fino a 50 microlitri. Questi volumi possono cambiare in base al protocollo di PCR utilizzato. </li><li> Mescolare delicatamente la reazione. Se necessario, raccogliere tutto il liquido sul fondo della provetta da un giro veloce. </li><li> Sovrapposizione del campione con olio minerale se si utilizza un termociclatore senza coperchio riscaldato. </li><li> Trasferire i tubi PCR dal ghiaccio ad un termociclatore con il blocco preriscaldato a 94 gradi Celsius e avviare il programma di ciclismo. Per una routine 3-step PCR, ci dovrebbe essere un passo denaturazione iniziale a 94 ° C per 30 secondi, seguita da 30 cicli di 94 gradi Celsius denaturazione per 15 secondi, da 55 a 65 gradi Celsius ricottura per 30 secondi, e 65 gradi Celsius estensione per 20 secondi, o 50 secondi per kb. Questo dovrebbe essere seguito da una estensione finale a 65 ° C per 5 minuti. Real-time PCR può essere eseguita anche per quantificare quantità di 5-metilcitosina e 5-hydroxymethylcytosine. Si prega di fare riferimento al manuale del prodotto, che si trova sulla neb.com per informazioni specifiche. </li></ol> 4. Rappresentante dei risultati: <img src="/files/ftp_upload/2661/2661fig1.jpg" alt="Figura 1" /> Figura 1. Confronto di 5-hydroxymethylcytosine ammonta a 12 nel locus diversi topo Balb / C campioni di tessuto. (A), End-point PCR. (B), Real time PCR. DNA da quattro tessuti del mouse è stato analizzato. Ai fini comparativi, PCR in tempo reale i dati sono stati normalizzati al DNA non tagliato. Una curva standard è stato utilizzato per determinare il numero di copia. I campioni potrebbero essere normalizzati dividendo il numero di copie di campioni Nessun 1-6 dal numero di copie del controllo che viene digerito (n. 5). Corsia gel Boxed mostra variazione in 5-HMC presente.

<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>

Sia Ted e rom, autori di questo articolo, sono dipendenti della ONA; rom è lo sviluppatore principale del kit, con Ted è il Direttore del Dipartimento Applicazioni &amp; Sviluppo che ha supervisionato lo sviluppo di questo kit.

Ci sono parecchie cose fondamentali da considerare nella creazione di questo esperimento. In primo luogo, è importante che il glucosylation di DNA genomico procede al completamento. La specificità sequenza di T4-BGT non è nota, e sembra non avere altra scelta per la sequenza di substrato. Pertanto, in alcuni casi, i tempi di incubazione più lungo può essere necessario. In secondo luogo la digestione, MspI e HpaII deve essere completa in modo da evitare segnale di fondo. Per questi enzimi di restrizione, si consiglia un tempo di incubazione di 4 ore, ma più incubazioni può essere eseguita quando scissione incompleta è osservato. In terzo luogo, il DNA quantità di ingresso può essere regolata a seconda della disponibilità, dal 20 NGS del DNA può essere usato per end-point PCR. Infine, si consiglia l&#39;uso del DNA di controllo in dotazione, che può essere eseguito in parallelo con il DNA genomico per 5 e 5-MC-HMC quantificazione.

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		<th>Material Name</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>
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<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

Hydroxymethylation DNA è una modificazione del DNA da tempo, ma recentemente è diventato un focus di ricerca epigenetica. DNA dei mammiferi è enzimaticamente modificati nella posizione di carbonio 5 ° della citosina (C) residui a 5-MC, prevalentemente nel contesto di dinucleotidi CpG. 5-MC è suscettibile di ossidazione enzimatica a 5-HMC dalla famiglia Tet di enzimi, che si ritiene siano coinvolti nello sviluppo e nella malattia. Attualmente, il ruolo biologico di 5-HMC non è pienamente compreso, ma sta generando molto interesse a causa della sua potenzialità come biomarker. Ciò è dovuto a diversi studi innovativi di identificazione 5-hydroxymethylcytosine nel topo staminali embrionali (ES) e le cellule neuronali. Tecniche di ricerca, compresi i metodi di sequenziamento bisolfito, non sono in grado di distinguere facilmente tra i 5-MC e 5-HMC. Un protocolli esistono pochi in grado di misurare quantità globale di 5-hydroxymethylcytosine nel genoma, tra cui la cromatografia liquida accoppiata con l&#39;analisi di spettrometria di massa e cromatografia su strato sottile di nucleosidi singolo digerito dal DNA genomico. Gli anticorpi che colpiscono 5-hydroxymethylcytosine esistono anche, che può essere utilizzato per l&#39;analisi dot blot, immunofluorescenza, o precipitazione del DNA hydroxymethylated, ma questi anticorpi non hanno singola aggiunta resolution.In di base, la risoluzione dipende dalle dimensioni del DNA immunoprecipitato e per esperimenti di microarray, dipende dal disegno della sonda. Dal momento che non si sa esattamente dove 5-hydroxymethylcytosine esiste nel genoma o il suo ruolo nella regolazione epigenetica, nuove tecniche, sono necessari in grado di identificare hydroxymethylation specifico locus. Il EpiMark 5-HMC e 5-mC Analisi Kit offre una soluzione di distinzione tra questi due modifiche a loci specifici. Il EpiMark 5-HMC e 5-mC Analisi Kit è un metodo semplice e robusto per l&#39;identificazione e quantificazione di 5-metilcitosina e 5-hydroxymethylcytosine all&#39;interno di un locus DNA specifico. Questo approccio enzimatica utilizza la sensibilità metilazione differenziale dei MspI isoschizomers e HpaII in un semplice 3-step del protocollo. DNA genomico di interesse è trattato con T4-BGT, aggiungendo un moeity glucosio al 5-hydroxymethylcytosine. Questa reazione è sequenza-indipendente, quindi tutti i 5-HMC sarà glucosilati, non modificato o 5-mC DNA contenente non saranno interessati. Questo glucosylation è poi seguita dalla digestione di restrizione endonucleasi. MspI e HpaII riconoscere la stessa sequenza (CCGG), ma sono sensibili a diversi stati di metilazione. HpaII si unirà solo un sito completamente modificato: ogni modifica (5-mC, 5-HMC o 5-ghmC) sia a scissione citosina blocchi. MspI riconosce e si unirà a 5 mC e 5-HMC, ma non 5-ghmC. La terza parte del protocollo è interrogatorio del locus mediante PCR. Appena 20 ng di DNA di ingresso possono essere utilizzati. Amplificazione della sperimentazione (glucosilati e digerito) e di controllo (finto glucosilati e digerito) DNA bersaglio con primer che fiancheggiano un sito CCGG di interesse (100-200 bp) viene eseguita. Se il sito CpG contiene 5-hydroxymethylcytosine, una band viene rilevato dopo glucosylation e la digestione, ma non nel non-glucosilati reazione di controllo. PCR in tempo reale darà un&#39;approssimazione di quanto hydroxymethylcytosine è in questo particolare sito. In questo esperimento, analizzeremo il 5-hydroxymethylcytosine quantità in un Babl / C campione di cervello di topo per punto PCR finale.

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

Il EpiMark 5-HMC e 5-mC Kit analisi può essere utilizzato per analizzare e quantificare 5-metilcitosina e 5-hydroxymethylcytosine all&#39;interno di un luogo spe specifico. Il kit distingue 5-5-mC da HMC con l&#39;aggiunta di glucosio al gruppo ossidrilico di 5-HMC tramite una reazione enzimatica che utilizza β-glucosiltransferasi (T4-BGT). Quando 5-HMC si verifica nel contesto di CCGG, questa modifica converte un sito cleavable MspI a un non-cleavable sito.

High Sensitivity 5-hydroxymethylcytosine rilevamento in Balb / C tessuto cerebrale

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 Views.  New England Biolabs. Il EpiMark 5-HMC e 5-mC Kit analisi può essere utilizzato per analizzare e quantificare 5-metilcitosina e 5-hydroxymethylcytosine all'interno di un luogo spe specifico. Il kit distingue 5-5-mC da HMC con l'aggiunta di glucosio al gruppo ossidrilico di 5-HMC tramite una reazione enzimatica che utilizza β-glucosiltransferasi (T4-BGT). Quando 5-HMC si verifica nel contesto di CCGG, questa modifica converte un sito cleavable MspI a un non-cleavable sito.

Video: 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.

Rilevamento di palmitoilazione proteina in colture di neuroni ippocampali di immunoprecipitazione e di acil-Biotina Exchange (ABE)

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

Ricerca

Neuroscienze

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).

Analisi Metabolomica di cervello di ratto da Alta Risoluzione Spettroscopia di Risonanza Magnetica Nucleare di estratti di tessuto

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.

Usando fluorescenza cellulare attivo Ordinamento per esaminare Cell-Type-specifica espressione genica nel cervello di ratto tessuto

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.

Un metodo semplice e poco costoso per la determinazione fredda sensibilità e adattamento nei topi

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.

Rilevazione di assoni localizzato mRNA nelle sezioni del cervello Utilizzando ad alta risoluzione<em&gt; In Situ</em&gt; Ibridazione

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.

Sublimazione di DAN Matrix per la rilevazione e visualizzazione dei gangliosidi nel cervello di ratto Tissue per 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.

High-throughput analisi del comportamento motorio nell'analisi di Isola della drosofila

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.

Imaging 3D ad alta risoluzione dell'infezione da virus della rabbia nel tessuto cerebrale con la rimozione del solvente

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.

Utilizzo della fluorescenza Near-Infrared e scansione ad alta risoluzione per misurare l'espressione proteica nel cervello del roditore

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.

Il rilevamento di 5-Hydroxytethylcytosine in cellule staminali neurali e cervelli di topi

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