This work is supported by a grant from the Natural Sciences and Engineering Research Council of Canada (NSERC: 418391-2012). AC receives a scholarship from the Centre th&#233;matique de recherche en Neurosciences (CTRN). HDB is funded by a scholarship from the Fonds de Recherche en Sant&#233; du Qu&#233;bec (FRSQ) and ML is a FRSQ Chercheur-Boursier.

注:该实验是按照加拿大指南实验动物的护理和使用进行，并经拉瓦尔大学动物保护委员会。 1.样品制备注意:在这个例子中，我们使用鼠标大脑在出生后第1天。 <ol><li>使用低温麻醉在碎冰为3至4分钟为幼仔，然后在冰冷的L15培养基中尽可能快地解剖大脑。执行在2分钟内该步骤。 </li><li>在嵌入模具（物资比照列表）转让解剖大脑和填充照顾以定向标本所需的位置冰冻组织包埋媒体。在-80℃下在液态氮和存储立即冻结标本。执行在30秒内这一步。 注:样品可以在不损害RNA的质量被储存几个月。 </li></ol> 2.切片<ol><li>治疗膜COA泰德玻片（ 比照材料清单）与RNA酶表面净化解决方案，以消除RNA酶的任何踪迹。在DEPC水冲洗的幻灯片，让他们干。可替代地，烘烤幻灯片过夜，在200℃。 </li><li>在转让之前与表面去污RNA酶溶液清洗低温恒温器冷冻样品。设置低温恒温室的温度在-20℃和切片标本在10微米厚。收集组织切片上的膜涂层的幻灯片，让他们干10分钟染色前。可替代地，存储幻灯片在-80℃直至染色。 </li></ol> 3.准备染色解决方案<ol><li>准备所有解决方案只是在实验开始前，并使用在所有染色溶液RNA酶抑制剂（除洗涤溶液），以防止RNA降解。 </li><li>制备缓冲溶液。使用在所有的解决方案同样的缓冲液（无RNA酶的磷酸盐缓冲盐水（PBS组成），补充1％的BSA，0.2％的Triton和2％RNA酶抑制剂）。对于每个幻灯片，准备400微升缓冲液（200微升用于初级和200微升的二次抗体）。 </li><li>制备固定剂溶液:用70％的乙醇保持在-20℃。保持组织固定过程最少，以避免急剧减少，在RNA提取收率。 注意:可替换地，可使用补充的5％乙酸的95％的乙醇溶液保持在-20℃。 </li><li>通过在缓冲溶液稀释的第一抗体制备第一抗体溶液。为多巴胺能神经元的标记，可使用靶向酪氨酸羟化酶（TH，速率限制酶对多巴胺生产）的抗体。使用高浓度的抗体，以补偿快速孵育时间。使用TH抗体在稀释1:25。 注:在正常免疫协议，该抗体给出的稀释度1孵育过夜，当强信号:1000。在这个协议中，它是用来40X更concentrat由于短的孵育时间编由于高浓度的抗体用于此方法中，为了检查非特异性标记的任何增加执行标准的免疫染色并联。 </li><li>通过在缓冲溶液稀释的二级抗体制备二级抗体溶液。使用生物素化的第二抗体以1:1的浓度:100。 注意:在这个例子中，我们使用抗兔生物素化的抗体。 </li><li>制备的抗生物素蛋白 - 生物素复合物（ABC）对生物素化的二级抗体的检测溶液。使ABC溶液通过将化合物A和化合物B以1:1的浓度:100稀释于DEPC PBS中补充有2％的RNA酶抑制剂。将其添加到幻灯片之前制备抗生物素蛋白 - 生物素复合物30分钟。 </li></ol> 4.染色<ol><li>通过快速轻弹它们在空气中解冻，在从-80时℃的一个或两个幻灯片（或使用鼓风机）构成。用疏水性的环绕部分屏障笔，让干燥的几秒钟。 <ol><li>把载玻片在冷的固定剂溶液不超过5分钟，在-20℃下。摇一次在空气中，以除去过量的固定剂溶液然后，迅速浸载玻片于DEPC PBS 6-8次，洗涤。 </li><li>轻弹幻灯片以去除多余的DEPC PBS的。确保幻灯片除霜步骤不超过5分钟。 </li></ol></li><li>在一个干净的托盘处的幻灯片（预处理用表面RNA酶去污溶液，以消除RNA酶的任何迹线），并把200μl的初级抗体溶液的每个滑板（兔抗TH）上，在室温下10分钟。浸迅速滑动于DEPC PBS中3次，每次洗涤和轻弹载玻片一次以除去过量的DEPC PBS中。 </li><li>把200μl的第二抗体溶液的每张幻灯片上为6分钟，在室温下进行。浸迅速滑动3倍于DEPC PBS中洗涤并轻弹载玻片以除去过量的DEPC PBS中。 </li><li>把200微升的ABC的SOLU灰每张幻灯片在室温下4分钟上。浸迅速滑动于DEPC PBS中3次，每次洗涤和轻弹载玻片以除去过量的DEPC PBS中。 </li><li>准备DAB（3,3&#39;二氨基联苯胺）培养的ABC解的4分钟内解决。使用在试剂盒（DAB过氧化物酶底物试剂盒）提供的试剂。拌1滴提供缓冲液中，1滴DAB溶液和1滴30％的H 2 O 2中的DEPC水2.5毫升。通过反转混合溶液。 </li><li>取196微升的DAB解决方案，只是在幻灯片上蔓延之前添加的RNA酶抑制剂的解决方案4微升（2％）。在一个干净的托盘（预处理表面RNA酶净化解决方案）的地方幻灯片，把200微升的DAB解决方案的幻灯片。 注:DAB的解决方案应该反应within1或2分钟。 </li><li>浸迅速滑动于DEPC PBS中3次，每次洗涤和放幻灯片几秒钟的无水乙醇。通过轻弹它们在空气干燥的幻灯片（或使用鼓风机）。&lt;BR /&gt;注:幻灯片可以储存在-80°C，以备将来使用或处理的LCM。 </li></ol> 5.激光捕获显微切割<ol><li>通过轻弹它们在空气中快速地解冻幻灯片（或使用鼓风机）构成。确保幻灯片除霜步骤不超过5分钟。 </li><li>进到LCM。放置幻灯片与面向收集管帽的样本面，在显微镜下。选择适合样品的最佳放大倍率。调节焦距以查看感兴趣的细胞。 <ol><li>限定在切割区域，并开始用激光切割。收集解剖的细胞或组织片在收集管帽填充有50微升裂解缓冲液（从RNA提取试剂盒）。确保LCM的步骤不超过30分钟。 </li></ol></li><li>提取使用RNA提取试剂盒（ 比照物料清单）分离细胞的总RNA。按照制造商的协议。 <ol><li>在42℃孵育收集的细胞进行30分钟至裂解细胞。先决条件，加入250微升调理缓冲器的RNA纯化柱。负载50微升乙醇溶解的细胞。加载100微升裂解的细胞到一个预条件纯化塔。 </li><li>离心柱在16000×g离心，在室温下2分钟。用水洗两次缓冲洗柱。洗脱在无RNA酶的水的最小推荐量（11微升）（未DEPC处理，因为它可能与生物分析器干扰）。保持2微升到测试质量。 注:所有的解决方案和试剂的试剂盒中提供。 </li></ol></li></ol> 6. cDNA合成和定量RT-PCR <ol><li>反向转录使用逆转录酶和寡（dT）的RNA样品从激光捕获多巴胺能细胞分化成互补DNA。按照制造商的协议（ 比照材料清单）。 </li><li>使用1微升cDNA的定量RT-PCR扩增与基因特异性引物。建立PCR意图在20μl体积与单组分热启动反应混合物用于定量PCR ctions如制造商推荐的。进行一式三份的每个反应中。 <ol><li>对于这里描述的实验中，使用以下引物对:GAPDH-F（5&#39;-CCA CCC AGA AGA CTG TGG的AT-3&#39;）/ GAPDH-R（5&#39;-GGA TGC AGG GAT GAT GTT CT-3&#39;）; RPL13A-F（5&#39;-ACA GCC ACT CTG GAG GAG AA-3&#39;）/ RPL13A-R（5&#39;-CTG C​​CT GTT TCC GTA ACC TC-3&#39;）; TH-F（5&#39;-CAG TGG AGG ATG TGT CTC-3&#39;）/钍-R（5&#39;-GAA AAT CAC GGG CAG ACA G-3&#39;）; AADC-F（5&#39;-CAT GAG AGC TTC TGC CCT TC -3&#39;）/ AADC-R（5&#39;-GGA TGT GGT CCC CAG TGT AG -3&#39;）。 </li></ol></li><li>使用以下的快速循环参数进行定量RT-PCR扩增:95℃下5分钟，随后10秒的45个循环95℃，10秒，在60℃，10秒72℃。使用仪器的默认设置determi进行熔解曲线分析NE同质产品的形成。 </li><li>分析使用内置的软件中的数据。 <ol><li>正常化的DA标记基因的Th和AADC的表情值与这两个参考基因GAPDH和RPL13A的表达，以获得相对表达水平如前面14所描述。 </li></ol></li></ol>

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作者什么都没有透露。

这种方法的最关键的一点在于，同时防止RNA降解标记所关注的细胞。如RNA酶是活性的水溶液，减少温育时间提高的RNA保护11,15。所用的所有解决方案必须用DEPC灭活RNA酶处理。所有的材料和表面需要与表面RNA酶去污溶液，以防止RNA酶污染处理。最后，在这些条件下，在所有的溶液中加入RNA酶抑制剂是有效的，从而降低维护的RNA。在使用过程中抗体孵育施加高盐条件先前已经证明是有效的在?...

大脑是由大量不同形成的复杂网络的不同神经元类型。这些神经元根据其形态，连接性和基因表达模式1组织在不同的组和子组。微阵列的发展，新一代测序，和定量RT-PCR技术提供给在不同的生物的上下文1,2-比较神经元群体的基因表达图谱的可能性。这些敏感分析需要的利益，同时保持RNA的完整性2-4细胞类型的精确鉴定和分离。荧光激活细胞分选（FACS）技术已被广泛地用于分离根据细胞表面标志物和/或形态的特定细胞类型。流式细胞仪需要一个细胞解离步骤之前进行排序，这导致空间分辨率5的完全丢失。许多神经元的亚群是彼此可分辨根据它们在第解剖学分布Ë大脑。施加薄的脑切片的激光捕获显微切割提供了用于细胞以高空间分辨率6-8的特定隔离的适当选项。 LCM的一个主要限制是需要确定基于形态学标准或荧光蛋白记者基因工程在动物模型中所关注的细胞。新技术的发展，以执行保存RNA的完整性，与LCM结合快速免疫标记方法，现在允许细胞亚群的隔离进行基因分析实验。 这一方法已被预先执行，并且在几个出版物1,9-13简单说明。在这里，我们证明了详细的程序通过组合快速免疫标记与LCM获得高品质的RNA的细胞的一个复杂的组织结构的特定子集。我们将展示如何执行关键的关键步骤，以获得最大的RNA恢复，避免RNA降解，因为它可能标志贴着地影响基因表达分析。 对于该协议的示范，从一个天大的小鼠中脑多巴胺能神经元的目标。多巴胺能神经元可以使用针对酪氨酸羟化酶（TH）抗体，对多巴胺合成的限速酶免疫标记。以下TH免疫染色，从个人或多巴胺能神经元的组然后可以使用LCM隔离。显微细胞被收集在裂解缓冲液，和RNA是使用RNA分离试剂盒提取。质量和提取RNA的量使用的是生物分析仪测量5。随后可以进行定量RT-PCR检测RNA测序，微阵列，或2,4,6-:使用基因表达的进一步分析。作为一个例子，一个两步QRT-PCR证明在激光捕获分离多巴胺能域。两个多巴胺能神经元标记基因的表达水平相对定量说明了这一协议的选择性。

<table><tbody><tr><td>Membrane coated slides&amp;nbsp;</td><td>Leica Biosystems</td><td>11505158</td><td>MembraneSlides PEN-Membrane 2,0 &amp;micro;m; 50 pcs</td></tr><tr><td>Surface RNAse decontamination solution</td><td>Life technologies</td><td>AM9780</td><td>RNaseZap</td></tr><tr><td>Fixative</td><td></td><td></td><td>70% Ethanol kept at -20 &amp;deg;C</td></tr><tr><td>Anti-TH&amp;nbsp;</td><td>Pel-Freez</td><td>P40101</td><td>&amp;nbsp;1:25</td></tr><tr><td>RNAse free buffer</td><td></td><td></td><td>DEPC PBS + 1% BSA+0.02% triton</td></tr><tr><td>RNAse inhibitor</td><td>Promega</td><td>N2615</td><td>Rnasine Plus Rnase Inhibitor 40 kU/ml (stock)</td></tr><tr><td>Anti-rabbit biotinylated</td><td>Vector Laboratories</td><td>BA-1000</td><td></td></tr><tr><td>Vectastain Elite ABC kit Standard</td><td>Vector Laboratories</td><td>PK-6100</td><td>8&amp;micro;l/ml</td></tr><tr><td>DAB Peroxidase Substrate Kit&amp;nbsp;</td><td>Vector Laboratories</td><td>SK-4100</td><td></td></tr><tr><td>RNA isolation kit</td><td>Life technologies</td><td>KIT0204</td><td>Arcturus PicoPure RNA Isolation Kit</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; 30%</td><td>Sigma</td><td>HC4060</td><td></td></tr><tr><td>Ethanol absolute</td><td></td><td></td><td></td></tr><tr><td>Laser microdissection system</td><td>Leica microsystems</td><td></td><td>Model AS-LMD</td></tr><tr><td>DEPC</td><td>Alfa Aesar</td><td>B22753-14</td><td></td></tr><tr><td>Bioanalyzer</td><td>Agilent</td><td></td><td>Agilent 2100 Bioanalyzer</td></tr><tr><td>Embedding mold&amp;nbsp;</td><td>Leica Biosystems</td><td>14702218311</td><td>6x8mm</td></tr><tr><td>Hydrophobic barrier pen&amp;nbsp;</td><td>Vector Laboratories</td><td>H-4000</td><td></td></tr><tr><td>Frozen tissue embedding media</td><td>Tissue-Tek</td><td>4583</td><td></td></tr><tr><td>Bioanalyzer chip</td><td>Aligent Technologies</td><td>5067-1513</td><td>RNA 6000 Pico LabChip used with Agilent 2100 Bioanalyzer</td></tr><tr><td>Hot start reaction mix for quantitative PCR</td><td>Roche</td><td>6402712001</td><td>Fast Start Essential DNA Green Master</td></tr><tr><td>Reverse transcriptase</td><td>Life technologies</td><td>11752-050</td><td>SuperScript III First-Strand Synthesis SuperMix for qRT-PCR</td></tr></tbody></table>

rna isolation from cell specific subpopulations using laser-capture microdissection combined with rapid immunolabeling

激光捕获显微切割（LCM）允许从薄的组织切片以高空间分辨率的特定细胞的分离。有效的LCM要求从异质组织精确识别的细胞亚群。对于LCM感兴趣细胞的识别通常是基于形态学标准或荧光蛋白记者。 LCM和快速的免疫标记的组合提供了可视化特定的细胞类型，并从周围组织分离它们的替代性和有效的手段。高质量的RNA随后可以从一个纯的细胞群中提取，并进一步处理用于下游应用，包括RNA测序，微阵列或QRT-PCR。这一方法已被预先执行，并且在几个出版物简要描述。本文的目的是要说明如何执行的细胞群体的快速免疫标记，同时保持RNA的完整性，以及如何分离使用LCM这些特定细胞。在此，我们举例说明D由免疫标记，并从1日龄的小鼠采集的多巴胺能细胞的脑组织在该多步骤过程。我们强调的是特别值得考虑的关键的关键步骤。此协议可适应各种组织和感兴趣的细胞。来自不同领域的研究人员将有可能从这种方法的演示中受益。

这种快速免疫标记过程允许同时保持RNA的完整性的关注的细胞可视化。 图1示出的RNA提取前组织制剂的步骤。 cryosectioning后，多巴胺能神经元使用针对酪氨酸羟化酶的抗体（标记的神经元出现在棕色）标记。根据实验问题，单个细胞或更大的感兴趣区域可以使用LCM被隔离（ 图1D - E）。它评估提取的RNA的质量，因为降解的RNA会对以下分析的质量相当大的影响是?...

Watch this Scientific Journal Video about RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling at JoVE.com

RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

细胞在特定组织的一个子集的基因表达分析是一个重大的挑战。本文介绍了如何通过将快速免疫标记方法与激光捕获显微切割分离，从一个特定的细胞群高质量的总RNA。

从细胞亚群特异性RNA提取使用激光捕获显微切割加上快速的免疫标记

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires ...

rna-isolation-from-cell-specific-subpopulations-using-laser-capture

Research

JoVE Journal

Biology

12.4K 次观看.  Université Laval. 细胞在特定组织的一个子集的基因表达分析是一个重大的挑战。本文介绍了如何通过将快速免疫标记方法与激光捕获显微切割分离，从一个特定的细胞群高质量的总RNA。 

视频: RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell activation, stress, or stimulation. Single-cell multiplex gene expression enables the simultaneous assessment of tens of genes1,2,3. At the single-cell level, multiplex gene expression determines population heterogeneity4,5. It allows for the distinction of population heterogeneity by determining both the probable mix of diverse precursor stages among mature cells and also the diversity of cell responses to stimuli.
Innate lymphoid cells (ILC) have been recently described as a population of innate effectors of the immune response6,7. In this protocol, cell heterogeneity of the ILC hepatic compartment is investigated during homeostasis.
Currently, the most widely used technique to assess gene expression is RT-qPCR. This method measures gene expression only one gene at a time. Additionally, this method cannot estimate heterogeneity of gene expression, since multiple cells are needed for one test. This leads to the measurement of the average gene expression of the population. When assessing large numbers of genes, RT-qPCR becomes a time-, reagent-, and sample-consuming method. Hence, the trade-offs limit the number of genes or cell populations that can be evaluated, increasing the risk of missing the global picture.
This manuscript describes how single-cell multiplex RT-qPCR can be used to overcome these limitations. This technique has benefited from recent microfluidics technological advances1,2. Reactions occurring in multiplex RT-qPCR chips do not exceed the nanoliter-level. Hence, single-cell gene expression, as well as simultaneous multiple gene expression, can be performed in a reagent-, sample-, and cost-effective manner. It is possible to test cell gene signature heterogeneity at the clonal level between cell subsets within a population at different developmental stages or under different conditions4,5. Working on rare populations with large numbers of conditions at the single-cell level is no longer a restriction.

This protocol describes how to assess the expression of a large array of genes at the clonal level. Single-cell RT-qPCR produces highly reliable results with a strong sensitivity for hundreds of samples and genes.

单细胞基因表达利用多重RT-qPCR的表征罕见的淋巴人群的异质性

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell ...

科研

遗传学

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

激光捕获显微切割技术的果蝇外周神经元

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

神经科学

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

激光捕获显微切割哺乳动物组织

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

生物学

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

激光捕获显微切割的丰富种群的创伤性脑损伤后神经元或单个神经元的基因表达分析

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) to provide an abundant supply of cells. NPCs were differentiated in the presence of a new combination of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), dibutyryl cAMP (DBC) and retinoic acid on dishes coated with poly-L-lysine and mouse laminin to obtain neuron-rich cultures. NPCs were also differentiated in the absence of neurotrophins, DBC and retinoic acid and in the presence of ciliary neurotrophic factor (CNTF) to yield astrocyte-rich cultures. Differentiated NPCs were characterized by immunofluorescence staining for a panel of neuronal markers including NeuN, synapsin, acetylcholinesterase, synaptophysin and GAP43. Glial fibrillary acidic protein (GFAP) and STAT3, astrocyte markers, were detected in 10-15% of differentiated NPCs. To facilitate cell-type specific molecular characterization, laser capture microdissection was performed to isolate neurons cultured on polyethylene naphthalate (PEN) membrane slides. The methods described in this study provide valuable tools to advance our understanding of the molecular mechanism of neurodegeneration.

Human neuroprogenitor cells (NPCs) were expanded under proliferating conditions. NPCs were differentiated into neuron-rich cultures in the presence of a combination of neurotrophins. Neuronal markers were detected by immunofluorescence staining. To isolate a pure population of neurons, NPCs were differentiated on PEN membrane slides and laser capture microdissection was performed.

神经元的分化的人类Neuroprogenitor细胞培养激光捕获显微切割

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

激光捕获显微切割 - 个人多巴胺神经元的分离和整个腹侧被盖区的示范

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive human diseases. Considerable advances have been made to uncover the molecular signatures of homeostatic microglia as well as alterations of their gene expression in response to environmental stimuli. With the advent and maturation of single-cell genomic methodologies, it is increasingly recognized that heterogenous microglia may underlie the diverse roles they play in different developmental and pathological conditions. Further dissection of such heterogeneity can be achieved through efficient isolation of microglia from a given region of interest, followed by sensitive profiling of individual cells. Here, we provide a detailed protocol for the rapid isolation of microglia from different brain regions in a single adult mouse brain hemisphere. We also demonstrate how to use these sorted microglia for plate-based deep single-cell RNA sequencing. We discuss the adaptability of this method to other scenarios and provide guidelines for improving the system to accommodate large-scale studies.

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

从一个成年小鼠脑半球分离区域特异性微胶质，进行深度单细胞RNA测序

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila tissues often contain a heterogeneous mixture of cell types.&#160;To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.

标记的细胞核从亲和力为基础的隔离<em&gt;果蝇</em&gt;组织的基因表达分析

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

捕获口腔粘膜下纤维化组织样本用于空间组学分析

Isolation of Cells with Morphological and Spatial Information from Oral Submucous Fibrosis Samples by Laser Capture Microdissection

Oral submucous fibrosis (OSF) is a common type of potentially malignant disorder in the oral cavity. The atrophy of epithelium and fibrosis of the lamina propria and the submucosa are often found on histopathological slides. Epithelial dysplasia, epithelial atrophy, and senescent fibroblasts have been proposed to be associated with the malignant transformation of OSF. However, because of the heterogeneity of potentially malignant oral disorders and oral squamous cell carcinoma, it is difficult to identify the specific molecular mechanisms of malignant transformation in OSF. Here, we present a method to obtain a small number of epithelial or mesenchymal cells carrying morphological data and spatial information by laser capture microdissection on formalin-fixed paraffin-embedded tissue slides. Using a microscope, we can precisely capture microscale (~500 cells) dysplastic or atrophic epithelial tissue and fibrotic subepithelial tissue. The extracted cells can be evaluated by genome or transcriptome sequencing to acquire genomic and transcriptomic data with morphological and spatial information. This approach removes the heterogeneity of bulk OSF tissue sequencing and the interference caused by cells in non-lesioned areas, allowing for precise spatial-omics analysis of OSF tissue.

作者聚焦:揭开口腔潜在恶性肿瘤的神秘面纱 

Laser capture microdissection of oral submucous fibrosis tissues allows for precise extraction of cells from histological regions of interest for the analysis of multi-omics data with morphological and spatial information.

激光捕获显微切割从口腔粘膜下纤维化样本中分离具有形态学和空间信息的细胞

Oral submucous fibrosis (OSF) is a common type of potentially malignant disorder in the oral cavity. The atrophy of epithelium and fibrosis of the lamina ...

从细胞亚群特异性RNA提取使用激光捕获显微切割加上快速的免疫标记

摘要

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此视频中的章节

单细胞基因表达利用多重RT-qPCR的表征罕见的淋巴人群的异质性

激光捕获显微切割技术的果蝇外周神经元

激光捕获显微切割哺乳动物组织

激光捕获显微切割的丰富种群的创伤性脑损伤后神经元或单个神经元的基因表达分析

神经元的分化的人类Neuroprogenitor细胞培养激光捕获显微切割

激光捕获显微切割 - 个人多巴胺神经元的分离和整个腹侧被盖区的示范

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