Sriharsa Pradhan、シャノンモーリーキニー、ハング慶チン、Ju​​rate Bitinaite、ゆう鄭、ピエールオリビエエステベ、Romualdas Vaisvila、スティーブンE.ヤコブセンの研究室、UCLA。この作品は、部分的にNIH 1R44GM095209 - 01によってサポートされていました。

1。 DNAのグルコシル化とコントロール反応<ol><li>氷の上に1.5ミリリットルの反応管に、ゲノムDNAの5〜10マイクログラム（30マイクログラム/ミリリットルの最終濃度）、UDP -グルコースの12.4マイクロリットル（80マイクロモルの最終濃度）、NEBuffer 4の31マイクロリットルを混ぜるとまでヌクレアーゼフリー水を310リットル〜310マイクロリットルの総容積をもたらす。 </li><li> 155マイクロリットルずつの2つのチューブにこの反応混合物を分割する。 </li><li>そしてあるチューブにT4 -β-グルコの30台、または3マイクロリットルを、追加します。静かにピペッティングでよく混ぜる。第2のチューブは、制御反応であるので、水3リットルを追加し、代わりにT4 - BGTの。 </li><li> T4 - BGTは、サンプル中の5 - hydroxymethylcytosineグループの水酸基にグルコースを加えるとなる時間の間に、12〜18時間37℃で、両方のチューブをインキュベートします。 </li></ol> 2。制限酵素消化<ol><li>ラベルthree 0.2ミリリットルのPCRストリップチューブ番号1〜3。それぞれに反応混合物のアリコート50μlの。 </li><li>ラベルthree 0.2ミリリットルのPCRストリップチューブ番号4〜6です。それぞれにコントロールの混合物のアリコート50μlの。 </li><li>チューブ1と4にMspI 100単位、または1マイクロリットルを、追加。静かにピペッティングでよく混ぜる。 </li><li>チューブ2と5にHpaIIの50台、または1マイクロリットルを、追加。静かにピペッティングでよく混ぜる。 </li><li>彼らはコントロールであるとして、チューブ3と6には何も追加しないでください。 </li><li>少なくとも4時間、37℃での全6チューブをインキュベートします。 </li><li>各チューブにプロテイナーゼKの1マイクロリットルを追加し、30分間40℃でインキュベートする。 </li><li> 10分間、摂氏95度でインキュベートしてプロテイナーゼKを不活性化する。 </li></ol> 3。 PCR /定量PCRによってDNAを分析するこのプロトコルは、うまく実行することが示されているエンドポイントPCR用NEBのLongAmp Taqを使用しています。他のPCRのプロトコルを代用することができます。 <ol><li> 5X LongAmp のTaq反応バッファー、10ミリモルのdNTP、10マイクロモルフォワードプライマーの1マイクロリットル、10マイクロモルリバースプライマーの1マイクロリットル、テンプレートの3マイクロリットルの1.5マイクロリットルの10マイクロリットル：氷の上で0.2ミリリットルのPCR反応チューブに以下のコンポーネントを追加します。前の手順で、LongAmp Taq DNA ポリメラーゼの1マイクロリットル、およびヌクレアーゼフリー水50リットルまでのDNA。これらのボリュームは、使用されるPCRのプロトコールにしたがって変更される可能性があります。 </li><li>穏やかに反応を混在させる。必要に応じて、迅速なスピンでチューブの底にすべての液体を集める。 </li><li>フタがなく、サーマルサイクラーを使用する場合はミネラルオイルでサンプルをオーバーレイ。 </li><li>摂氏94度に予熱したブロックと氷からサーマルサイクラーにPCRチューブを転送し、サイクリングプログラムを起動します。ルーチンの3ステップPCRには、15秒、30秒間、アニーリング摂氏55から65度の変性摂氏94度、および拡張子摂氏65度の30サイクルで30秒間、摂氏94 ° 1つの初期変性ステップがあるはずです20秒、またはkbあたり50秒。これは、5分間摂氏65度に一つの最終的な拡張子が続く必要がある。リアルタイムPCRは、5 - メチルシトシン、5 - hydroxymethylcytosineの量を定量するために実行することができます。具体的な情報については、neb.com上で見つかった、製品のマニュアルを参照してください。 </li></ol> 4。代表的な結果： <img src="/files/ftp_upload/2661/2661fig1.jpg" alt="図1" /> 図1。異なるマウスのBalb / C組織サンプルの遺伝子座12番5 - hydroxymethylcytosine量の比較。 （A）、エンドポイントPCR。 （B）、リアルタイムPCR。 fourマウス組織からのDNAを分析した。比較のため、リアルタイムPCRのデータがカットされていないDNAに正規化された。標準曲線は、コピー数を決定するために使用された。サンプルは、未消化されているコントロールのコピー数（なし5）でサンプルなし1〜6のコピー数を除算して正規化することができます。箱入りのゲルのレーンが5 HMCの現在の変化を示しています。

<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>

テッドとRomas両方は、この記事の著者は、NEBの従業員です。Romasは、テッドは、このキットの開発を監督するアプリケーションおよび開発部門のディレクターであることと、キットの主要な開発者です。

この実験を設定する際に考慮すべきいくつかの重要な点があります。最初に、ゲノムDNAのグルコシル化が完了するまで進行することが重要です。 T4 - BGTの配列特異性が知られており、それが基質配列のための選択の余地がないように見えることがありますされていません。したがって、いくつかのケースでは、より長いインキュベーション時間が必要になる場合があります。第二に、MspIとHpaII消化は、バックグラウンドシグナルを避けるために完全なものでなければなりません。これらの制限酵素のた​​めに、我々は4時間のインキュベーション時間をお勧めしますが、不完全な切断が観察されたときより長いインキュベーションを行うことができます。第三に、インプットDNA量はDNAの20 NGSがエンドポイントPCRに使用することができるため、可用性に応じて調整することができます。最後に、我々は5 - MCと5 - HMCの定量化のためのゲノムDNAを並列で実行されることがあります付属のコントロールDNAを使用することをお勧めします。

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		<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

DNAのhydroxymethylationは、DNAの長い知られている変更ですが、最近ではエピジェネティクス研究の焦点となっている。哺乳動物のDNAを酵素的に主にCpGジヌクレオチドの文脈において、5 - MCにシトシン（C）残基の5 番目の炭素の位置に変更されます。 5 - MCは、開発と疾患に関与すると考えられる酵素のテトの家族、5 - HMCへの酵素的酸化に適している。現在、5 - HMCの生物学的役割は十分に理解されていないが、バイオマーカーとしての可能性があるため、多くの関心を生成している。これは、マウス胚性幹（ES）と神経細胞で5 - hydroxymethylcytosineを識別するいくつかの画期的な研究によるものです。 亜硫酸シークエンシングの方法を含む研究の技術は、、簡単に5 - MCと5 - HMCを区別することはできません。いくつかのプロトコルは、質量分析またはゲノムDNAから消化単一のヌクレオシドの薄層クロマトグラフィーと組み合わせた液体クロマトグラフィーを含む、ゲノムの5 - hydroxymethylcytosineのグローバルな量を測定することができますが存在する。ターゲット5 - hydroxymethylcytosineもドットブロット分析、免疫蛍光、またはhydroxymethylated DNAの沈殿のために使用されるが、これらの抗体は、単一の基地resolution.Inの加算がない、解像度が免疫沈降されたDNAのサイズとするために依存することができる、存在している抗体マイクロアレイ実験には、プローブの設計に依存します。それが不明なので、5 hydroxymethylcytosineはゲノムやエピジェネティックな調節におけるその役割に存在する場所を正確に、新しい技術は、遺伝子座特異的hydroxymethylationを識別できる必要があります。 EpiMark 5 - HMCおよび5 - MCの分析キットは、特定の遺伝子座で、これら2つの変更を区別するためのソリューションを提供します。 EpiMark 5 - HMCおよび5 - MCの分析キットは、5 - メチルシトシンと特異的なDNAの遺伝子座内5 - hydroxymethylcytosineの同定と定量のためのシンプルかつ堅牢な方法です。この酵素的アプローチは、シンプルな3ステップのプロトコールでisoschizomers MspIとHpaIIの差のメチル化の感度を利用しています。 関心のゲノムDNAを5 - hydroxymethylcytosineにグルコースmoeityを追加、T4 - BGTで処理される。この反応は、順序に依存しない、したがって、すべての5 - HMCはglucosylatedされている、未修飾または5 - MCを含むDNAは影響を受けません。 このグルコシル化は、制限エンドヌクレアーゼ消化が続いている。 MspIとHpaIIは、同じシーケンス（CCGG）を認識しますが、異なるメチル化状態に敏感です。シトシンブロックの切断のいずれかですべての変更（5 - MC、5 - HMCまたは5 - ghmC）：HpaIIは完全無修正サイトを切断する。 MspIは5 - MCと5 - HMCではなく、5 ghmCを認識し切断する。 プロトコルの3番目の部分は、PCRによる遺伝子座の尋問です。入力のDNAのわずか20 ngのを使用することができます。実験的な（glucosylatedと消化）とコントロール（モックはglucosylatedと消化）興味のCCGG部位（100〜200 bp）を隣接するプライマーとターゲットDNAの増幅が行われます。のCpGサイトが5 hydroxymethylcytosineが含まれている場合は、バンドがグルコシル化と消化の後、ではなく、非glucosylatedコントロール反応で検出される。リアルタイムPCRはhydroxymethylcytosineは、この特定のサイトにどれだけの近似を与える。 この実験では、エンドポイントPCRによってマウスBabl / C脳のサンプルで5 hydroxymethylcytosine量を分析します。

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

EpiMark 5 - HMCおよび5 - MCの分析キットは、5 - メチルシトシンとSPE cific座内5 - hydroxymethylcytosineを分析し、定量するために使用することができます。キットには、（T4 - BGT）β-グルコシルトランスフェラーゼを利用した酵素反応を介して5 - HMCの水酸基にグルコースを添加することによって5 - HMCから5 - MCを区別します。 5 - HMCがCCGGのコンテキストで発生すると、この変更は、非切断部位に切断可能なMspIのサイトを変換します。

のBalb / Cの脳組織の高感度5 hydroxymethylcytosine検出

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 ビュー.  New England Biolabs. EpiMark 5 - HMCおよび5 - MCの分析キットは、5 - メチルシトシンとSPE cific座内5 - hydroxymethylcytosineを分析し、定量するために使用することができます。キットには、（T4 - BGT）β-グルコシルトランスフェラーゼを利用した酵素反応を介して5 - HMCの水酸基にグルコースを添加することによって5 - HMCから5 - MCを区別します。 5 - HMCがCCGGのコンテキストで発生すると?...

ビデオ: 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.

免疫沈降およびアシル-ビオチン所（阿部）による培養海馬神経細胞におけるタンパク質パルミトイル化の検出

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

神経科学

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).

組織抽出物の高分解能核磁気共鳴分光法によるラット脳のメタボローム解析

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.

マウスにおける低温感受性と適応を決定するためのシンプルで安価な方法

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.

高解像度を使用した脳切片における軸索のローカライズされたmRNAの検出<em&gt;その場での</em&gt;ハイブリダイゼーション

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.

MALDIイメージングマススペクトロメトリーのためのラット脳組織におけるガングリオシドの検出と可視化のためのDANマトリックスの昇華

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.

ショウジョウバエ島アッセイにおける歩行行動の高スループット解析

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.

溶媒クリア脳組織における狂犬病ウイルス感染の高解像度3Dイメージング

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.

げっ歯類の脳におけるタンパク質発現を測定するために近赤外蛍光と高解像度走査を用いること

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.

マウスの神経幹細胞と脳における5-ヒドロキシメチルシトシンの検出

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