Sriharsa普拉丹，香侬 - 莫雷金尼，恒庆钦Jurate Bitinaite，郑瑜，皮埃尔奥利维尔Esteve，Romualdas Vaisvila，史蒂芬五雅各布森的实验室，加州大学洛杉矶分校。部分支持这项工作是由美国国立卫生研究院1R44GM095209 - 01。

1。 DNA Glucosylation和控制反应<ol><li>在反应管上冰的1.5毫升，混合5-10微克的基因组DNA（终浓度为30微克/毫升），12.4微升UDP -葡萄糖（终浓度为80微摩尔），31 NEBuffer 4微升高达310微升无核酸酶水带来的总体积310微升。 </li><li>拆分成两个，每管155微升反应混合物。 </li><li>然后添加30个单位，或3微升的T4 -β-葡萄糖，一管。向上和向下轻轻吹打混合。第二管是控制反应，所以加水3微升，而不是T4 - BGT。 </li><li>孵育都管，在37度摄氏12至18小时，在此期间，T4 - BGT将增加葡萄糖的5 hydroxymethylcytosine组样品中的羟基组。 </li></ol> 2。限制性内切酶消化<ol><li>三个标签0.2毫升PCR条管1至3号。分装到每个反应混合物50 UL。 </li><li>三个标签0.2毫升PCR条管4至6号。 UL混合控制到每一个分装50。 </li><li>添加到试管1和4 100个单位，或MSPI 1微升。向上和向下轻轻吹打混合。 </li><li>添加到管2和5的50个单位，或HpaII 1微升。向上和向下轻轻吹打混合。 </li><li>不要添加任何管3和6，因为他们控制。 </li><li>至少4个小时，在37摄氏度孵育的所有6个管。 </li><li>添加到每管1微升的蛋白酶K孵育30分钟，摄氏40度。 </li><li>灭活蛋白酶K孵育10分钟，摄氏95度。 </li></ol> 3。分析DNA PCR / qPCR的该协议使用，NEB的LongAmp Taq酶终点PCR这已被证明表现良好。可以取代其他PCR检测。 <ol><li>在冰上0.2毫升PCR反应管：10微升5X LongAmp Taq酶反应缓冲液，1.5微升，10毫摩尔dNTPs，1微升10微摩尔正向引物，1微升10微摩尔的反向引物，3微升模板添加下列组件从前面的步骤，LongAmp Taq DNA聚合酶1微升，无核酸酶水至50微升的DNA。根据PCR的协议，用于这些卷可能会改变。 </li><li>轻轻地混合反应。如果有必要，收集所有液体的试管底部，由快速旋转。 </li><li>与矿物油覆盖的样本，没有加热的盖子，如果使用一个热循环。 </li><li> PCR管冰转移到一个热循环与预热到94摄氏度的块，并开始循环程序。例行3步PCR，应在94摄氏度的初始变性30秒，30个循环94度摄氏变性15秒，55至65度摄氏30秒退火，延伸度和65摄氏度的步骤20秒或50秒每KB。这之后，应在65度摄氏5分钟的最后一次延长。实时PCR也可以进行定量5 - 甲基胞嘧啶和5 hydroxymethylcytosine金额。请参考产品使用说明书，在neb.com发现特定的信息。 </li></ol> 4。代表性的成果： <img src="/files/ftp_upload/2661/2661fig1.jpg" alt="图1" /> 图1 5 hydroxymethylcytosine金额在12位点的比较不同的小鼠BALB / C组织样本。 （一），终点PCR扩增。 （二），实时PCR。 从四个小鼠组织DNA进行了分析。为便于比较，未切割DNA的实时PCR数据正常化。标准曲线来确定套数。未消化的控制的拷贝数（5）除以样品没有1-6的拷贝数的样品可以归。盒装凝胶泳道显示在5 HMC目前的变化。

<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>

泰德和吉卜赛人，这篇文章的作者，是NEB员工;罗姆人是该套件的主要开发者特德的应用与发展部，负责本试剂盒的发展署署长。

设立这个实验时，有几个关键的事情要考虑。首先，它是非常重要的基因组DNA glucosylation收益来完成。 T4 - BGT序列特异性是不为人所知，并且，它似乎已经为基材序列别无选择。因此，在某些情况下，潜伏期较长时间，可能是必要的。其次，MSPI和HpaII消化必须是完整的，以避免背景信号。对于这些限制性内切酶，我们推荐的4个小时的潜伏期的时间，但不完全卵裂是观察时，可以进行较长的孵化。第三，输入DNA量可以调整取决于可用性，自20农工商超市的DNA可用于终点PCR。最后，我们建议使用提供的对照DNA，这可能是基因组DNA的同时运行5 - MC和5 - HMC量化。

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<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羟甲基化是一个久负盛名的DNA修饰，但最近已成为表观遗传学研究的重点。哺乳动物的DNA酶修饰的5 个碳的位置，胞嘧啶（C）残留5 - MC，他们主要在CpG二核苷酸中。 5 - MC是服从酶氧化酶的春节家庭，这被认为是涉及发育和疾病5 - HMC。目前，5 HMC的生物作用是不完全理解，但产生了很多的利益，由于其作为生物标志物的潜力。这是由于几个开创性的研究确定，在小鼠胚胎干（ES）和神经细胞的5 - hydroxymethylcytosine。 研究技术，包括重亚硫酸盐测序方法，是无法容易分辨5 - MC和5 HMC。几个协议存在，可以衡量全球基因组中的5 - hydroxymethylcytosine的金额，包括液相色谱与质谱分析或薄层色谱法从基因组DNA消化的单核苷，的。抗体，目标5 - hydroxymethylcytosine也存在，可用于点杂交分析，免疫荧光，或羟甲基化的DNA沉淀，但这些抗体不具备单基地resolution.In此外，分辨率取决于免疫沉淀的DNA的大小和微阵列实验，取决于探头设计。由于它是未知的5 hydroxymethylcytosine在基因组的表观遗传调控中的作用存在的确切位置，新技术，它可以识别位点的具体羟甲基。 EpiMark 5 HMC和5 - MC分析套件提供了一个区分在特定位点的这两个修改的解决方案。 EpiMark 5 HMC和5 - MC分析套件是为5 - 甲基胞嘧啶和5 hydroxymethylcytosine在一个特定的DNA位点的识别和定量分析的简单和可靠的方法。这种酶的方法，利用同裂酶MSPI和HpaII差甲基化的敏感性，在一个简单的3步协议。 被视为与T4 - BGT感兴趣的基因组DNA，加入葡萄糖moeity 5 hydroxymethylcytosine。这种反应是序列独立的，因此所有5 - HMC将glucosylated;未修改或5 - MC含有DNA不会受到影响。 这glucosylation是随后由酶切。 MSPI和HpaII认识到相同的序列（CCGG），但不同的甲基化状态很敏感。 HpaII切割只有一个完全未修改的网站：任何修改或者胞嘧啶块裂解（5 - MC，5 HMC或5 ghmC）。 MSPI识别和切割5 - MC和5 HMC，但不是5 ghmC。 该协议的第三部分是通过PCR的轨迹审讯。可用于输入DNA低至20纳克。实验（glucosylated和消化）和控制（模拟glucosylated和消化）的目标与侧翼利益CCGG网站（100-200 BP）引物的DNA进行扩增。如果中央人民政府网站包含5 hydroxymethylcytosine，带检测glucosylation和消化后，但不能在非glucosylated控制反应。实时PCR将在这个特殊的网站是多少hydroxymethylcytosine逼近。 在这个实验中，我们将分析终点PCR方法在小鼠BABL / C大脑样本5 hydroxymethylcytosine金额。

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和5 - MC分析套件，可用于分析和定量内SPE太平洋轨迹的5 - 甲基胞嘧啶和5 hydroxymethylcytosine。该试剂盒区别于5 HMC 5 - MC除了通过对葡萄糖的酶，利用β-葡萄糖（T4 - BGT）反应羟基组的5 HMC。当5 HMC在CCGG背景下发生，这种修改裂解MSPI网站转换成非裂解网站。

BALB / C脑组织中的高灵敏度检测5 - hydroxymethylcytosine

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 次观看.  New England Biolabs. EpiMark 5 HMC和5 - MC分析套件，可用于分析和定量内SPE太平洋轨迹的5 - 甲基胞嘧啶和5 hydroxymethylcytosine。该试剂盒区别于5 HMC 5 - MC除了通过对葡萄糖的酶，利用β-葡萄糖（T4 - BGT）反应羟基组的5 HMC。当5 HMC在CCGG背景下发生，这种修改裂解MSPI网站转换成非裂解网站。

视频: 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.

检测蛋白质棕榈酰化培养海马神经元通过免疫沉淀和酰基-生物素交易所（ABE）

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

科研

神经科学

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).

大鼠脑组织被提取物的高分辨核磁共振代谢组学分析

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.

在大鼠脑组织用荧光活化细胞分选，以检查细胞类型特异性基因表达

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.

测定冷灵敏度和适应小鼠一种简单廉价的方法

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.

在脑切片检测轴突本地化mRNAs的高空间分辨率<em&gt;原位</em&gt;杂交

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.

DAN矩阵升华为神经节苷脂对大鼠脑组织的检测和可视化MALDI成像质谱

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.

果蝇岛试验中运动行为的高通量分析

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.

溶剂清除脑组织狂犬病病毒感染的高分辨率3D成像

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.

近红外荧光和高分辨率扫描测量啮齿类动物大脑中蛋白质的表达

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.

小鼠神经干细胞和脑中5-羟基甲基细胞氨酸的检测

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