This work was funded by the National Institute of Mental Health (1R15MH084209 - 01A1), National Institute of Neurological Disorders and Stroke (R21NS058375-01A2) and the National Science Foundation (DBI0723080 07070646).

注：从小鼠脑组织使用符合卫生指南实验动物的护理和使用的国家机构，并研究方案被批准的机构动物护理和使用委员会在密西西比州立大学。 1.准备幻灯片和组织<ol><li>请使用硅烷准备幻灯片或聚邻苯二甲酸酯（PEN）膜的载玻片上。 </li><li>对于RNA分离，在核糖核酸酶去污溶液浸幻灯片，洗3次，用无RNA酶的水，然后脱水以分级系列无RNase乙醇和真空干燥的30分钟。 </li><li>使用低温恒温器切割组织切片在10微米厚度和安装的准备核糖核酸免费的幻灯片。保持切片保持RNA的质量在冻结部分。 注：可以肯定的组织是约4mm距滑动的LCM不能除去组织过于接近滑动的边缘的边缘。在奥德r以从PEN膜滑动除去组织，确保该组织是从左侧，顶部和底部4毫米和7毫米的右侧分别是膜的未附连到滑动的尺寸的幻灯片。 </li><li>对于使用直接免疫组化，组织部分上任硅烷制备幻灯片或PEN膜幻灯片单个细胞群的隔离。使用间接免疫组织化学引导技术组织的区域隔离，安装一个套节上进行免疫组化和交替部分在任一的PEN膜滑动和/或硅烷制备型滑动硅烷制备型滑动用于LCM。 </li></ol> 2.组织准备激光捕获 - 快速酪氨酸羟化酶免疫的免疫活性细胞的直接激光捕获<ol><li>大纲组织与疏水笔并晾干。 </li><li>修复组织的丙酮 - 甲醇（1：1）溶液在-20℃下进行10分钟。 注：欧ř经验表明，丙酮 - 甲醇固定导致更一致的免疫比丙酮或甲醇单独。 </li><li>在磷酸盐冲洗滑动缓冲盐水（PBS），用1％的Triton（无RNA酶）。 </li><li>盖板部分与100-200微升的PBS，用1％的Triton与酪氨酸羟化酶抗体1：100稀释用400单位/毫升的RNasin。孵育5-10分钟。 </li><li>简要地在PBS中漂洗两次和PBS-1％的Triton。 </li><li>涵盖组织以100-200微升的山羊抗兔IgG标记的Alexa Fluor 488 1:100稀释在PBS-1％的Triton 400 U / ml的RNA酶抑制剂和50ng / ml的DAPI染色。孵育5分钟。 </li><li>漂洗2次在PBS中再脱水30秒在一个分级系列的无RNA酶的乙醇（75％-75％-95％-95％-100％-100％）。 </li><li>孵育在二甲苯洗涤两次，持续1分钟，然后5分钟。 </li><li>去除二甲苯幻灯片之前立即用于LCM，并允许风干。 </li></ol> 3.组织准备激光捕获 - 酪氨酸羟化酶免疫的免疫反应区的间接激光捕获<ol><li>过程一张幻灯片与相邻的部分路段安装在硅烷准备和/或PEN膜幻灯片进行免疫组化。 注意：此处所用的程序是类似于以前报道5。为示范的图像，所述二氨基联苯胺（DAB）发色反应物用于可视化感兴趣的酪氨酸羟化酶的神经元。 </li><li>对于用于LCM幻灯片，定为5分钟，在100％的丙酮在4℃下。 </li><li>脱水组织在分级一系列的RNase免乙醇（75％-75％-95％-95％-100％-100％），为每个1分钟。 </li><li>孵育在二甲苯洗涤两次，持续1分钟，然后5分钟。 </li><li>去除二甲苯幻灯片之前立即用于LCM，并允许风干。 </li></ol> 4.使用激光捕获显微切割系统注：LCM应用程序有10个工具条和2个窗口控制显微切割的过程。工具栏：1.显微镜，2捕获激光，3.激光切割4.研究，5.导航，6材料，7捕获组，8显微切割，9字体，10注解。 Windows系统：1.实时视频和2路线图。 <ol><li>选择在LCM系统软件的材料工具栏上的“开门”选项卡开门。 </li><li>负载幻灯片上的三个可用插槽。对于本演示中，载一张幻灯片与酪氨酸羟化酶免疫组化可视化与DAB，一是硅烷准备的幻灯片和一个PEN膜各打成快速荧光酪氨酸羟化酶免疫组化幻灯片。在典型的实验中，使用未标记的丙酮固定切片与标记为用DAB酪氨酸羟化酶或幻灯片直接标记荧光酪氨酸羟化酶用于激光捕获的参考幻灯片。 </li><li>加载LCM帽到可用插槽。 注：宏和HS LCM盖可供使用。使用LCM帽的类型代暂时搁置于细胞或组织的量的数目被收集。宏幻灯片列出收集多达几千细胞的同时HS帽可以收集几百个。宏幻灯片让更多的组织收集，但需要50微升的裂解液。在HS帽，与适配器一起使用时，只需要10微升裂解缓冲液，它允许用于RNA的更大比例的恢复。 </li><li>关门。 </li><li>加载滑激活路地图窗口，它提供了整个滑动的工作图，这允许选择感兴趣的区域的（ 图3）。查看实时视频窗口中的路线图，导航到感兴趣的区域。 </li><li>在材质的工具栏，通过检查上面的滑块槽框标识PEN膜的幻灯片。供应帽的属性，右键点击帽的材料工具栏，选择“帽供应的属性”。检查表示帽的位置的框，并选择任一马CRO或HS帽。 </li><li>选择在显微镜工具栏上的“荧光”标签开启荧光灯及使荧光灯预热。使用蓝色和紫外线过滤器，以可视化的亚历克斯福陆公司488和DAPI分别。用“照相机调整”标签来增加，以便看到荧光标记的细胞的图像的灵敏度。 </li><li>在材质的工具栏，右键单击一个上限，并将其移动到感兴趣的幻灯片。双击在路线图的某个位置选择出现在实时视频窗口的区域。通过选择路线图的区域（左键双击），然后右击并选择移动周围的上限幻灯片“在中心区域将帽。” </li><li>使用红外激光捕获前，瞄准和调整它的实力。放置在盖在滑动远离组织的区域。在捕获激光工具栏，选择“启用”。调整电源（3-100毫瓦），脉冲（100-1000000μ;秒）和＃命中激光。火IR激光器，当所述端盖上滑动，而不组织的一部分，并检查，以查看是否在激光充分地熔化LCM帽膜。 注意：这就是所谓的润湿被熔化聚合物膜在盖以便其接触载玻片。足够的润湿是可见的暗环稠合的环清除（参见图4的充不足熔点的例子）的中心滑动。如果润湿不充分，再次调整功率和脉冲，并测试火灾直到实现这一点。此外，也可以使用激光的多个探测器。 </li><li>当红外激光强度进行调整，点击右键，选择“捕获激光是在这里”瞄准激光。 注：此步骤告诉那里的红外激光捕获的目的是在组织的计算机。瞄准激光的这一步骤是非常关键的，并且需要每个盖被移动时要重复或目标被改变。 </li></oL&gt; 个人多巴胺神经元5.激光捕获显微切割掉硅烷准备幻灯片<ol><li>移动到感兴趣的区域，以捕获细胞。 注：分离多巴胺神经元的例子在这里示出。 </li><li>要捕获离硅烷准备幻灯片的单个细胞，选择“LCM”选项卡，然后在“单点”，在显微切割工具栏中的图标。 </li><li>在显微镜工具栏，使用“快门”标签荧光滤镜片荧光和明场之间进行切换，以选择合适的过滤器，并使用目标选项卡来改变倍率。一旦所感兴趣的细胞是在实时视频窗口中可见，调整用于所关注的细胞标记的细胞的大致尺寸的斑点尺寸。通过点击捕获激光工具栏然后点击一个染色细胞，然后通过点击在相反侧的一侧上的地点标签做到这一点。 注：此计算直径的光点，并显示该值。 </li><li>通过点击它们而“单点”的图标被选择标记感兴趣的细胞。这样做是为了把一个点标记每个单元上。在这里，蓝色过滤器和10X物镜被用于可视化的多巴胺神经元。重新测试和瞄准红外激光的上限那里按照步骤4.9和4.10描述的是它下面的任何组织。 </li><li>一旦细胞标记，请选择“进入 - 切割和捕获”显微切割工具栏和红外激光对标签会自动闪光选择的所有标记的​​细胞。此外，如果需要的话，手动触发的IR激光器的任何兴趣点（ 图5D）。 </li><li>移动盖远离部和上滑动的开口区域，以检查是否将细胞捕获在LCM的帽。确保感兴趣的细胞是从滑动（ 图5B中的E）并连接到LCM盖（ 图5C中的F）。文档日过程是通过单击显微镜工具栏中的“捕获图像”选项卡上。 </li><li>当收集完组织，去除帽帽右击并选择“移动到”，然后“卸载”。 </li></ol>个人多巴胺神经元6.激光捕获过PEN膜幻灯片<ol><li>捕捉到单个细胞脱落的同时使用红外和紫外激光器PEN膜的幻灯片，选择“剪切和捕捉”选项卡，在显微切割工具栏。选择“单点”标签来标记感兴趣的细胞。使用“剪切线”选项卡概述每个单元格。一定要测试的IR激光器如在步骤4.9和4.10中描述并测试UV激光，以确定认为必要削减最小强度在PEN膜和组织（ 图4和5） </li><li>选择显微切割工具栏上的“捕捉和剪切”选项卡。当使用这种技术收集个体细胞，是务必火IR激光器第一后跟UV激光，因为这些小片可以是当它们存在之前切出，以被固定到LCM盖丢失。 注意：此过程也可以通过烧制红外激光在兴趣点的手动完成并将细胞单独列出与紫外激光。 </li><li>收集感兴趣的细胞后，如在步骤5.7如上所述，以确定是否被拆除和附连到所述盖（ 图6）的细胞移动的LCM帽。 </li><li>当收集完组织，取下帽子用鼠标右键单击帽，选择“移动到”，然后“卸载”。 </li></ol> 7.硅烷准备幻灯片捕获的腹侧被盖区<ol><li>来识别感兴趣的区域，则使用处理用于免疫组织化学的幻灯片。 注：对于本演示腹侧被盖区被隔离（ 图7A）。 </li><li>加载LCM帽和测试激光作为步骤š4.9和4.10。 注意：通常情况下，一个宏LCM盖被使用，但一个协帽也可以使用（参见图8）。 </li><li>为了隔离组织从硅烷准备幻灯片的区域（ 即腹侧被盖区），请在显微切割工具栏中的“LCM”选项卡，然后选择“多边形外观”选项卡。概述在实时视频窗口感兴趣的区域。根据收集的区域的大小，勾勒出在低放大倍数（2X目的）感兴趣区域。收集，但是，仪器使用10倍的目标，以便调整目标下，10倍的目标的红外激光之前捕获。 </li><li>选择 - 在显微切割工具栏中的“去捕捉和切”选项卡。 注：计算机会自动闪光红外激光在整个所选区域（ 图7F，I）。如果液晶膜没有充分熔化整个区域中，激光可发射人ually或整个过程重复进行。 </li><li>移动LCM帽从组织，以确定所述组织是如何好收集。 注：使用这种方法的一个典型的通过留下一些组织背后的幻灯片（见Figure7G）。如果发生这种情况，则重复选择和上述捕获步骤。重复这一步骤可以增加组织的收集（ 图7J）的量。 </li><li>当收集完组织，卸帽通过右键点击帽，选择“移动到”，然后“卸载”。 </li></ol> 8.捕获从PEN膜幻灯片的腹侧被盖区<ol><li>使用处理用于免疫组织化学的滑动为指导，以从上在PEN膜滑动如上所述组织选择感兴趣的区域。 </li><li>选择在显微切割工具栏中的“剪切和捕捉”选项卡。选择“剪切线”选项卡，并使用免疫组勾勒出感兴趣区域米斯特里标记的幻灯片作为指导。 注：该程序会自动添加点的红外激光对组织附着到帽。使用“单点”选项卡中添加额外的点，以更好地保护组织的上限。 </li><li>根据10倍的目标，试射和在4.9和4.10描述瞄准红外和紫外激光。测试该UV激光可以穿透细胞膜上滑动，并且该切口对应于计算机屏幕上的位置。使用紫外激光的强度是刚好足以切割的膜和组织，但不损伤所述组织（ 图5）。 </li><li>选择 - 在显微切割工具栏中的“去捕捉和切”选项卡。 注：计算机会自动闪光的红外激光在组织中的所有标记点把它连接到盖那么火紫外激光切割幻灯片PEN膜和组织（ 图7C）。附加红外熔化点可能是必要的，以充分地附着在TIS告到LCM帽，这也可以手动添加。 </li><li>移动LCM帽从组织，以确定是否将组织从部分（ 图7C）除去和附连到所述盖（ 图7C）。 </li><li>当收集完组织，去除瓶盖，右键单击帽，然后选择“移动到”，然后“卸载”。 </li></ol> 9.隔离的RNA <ol><li>收集来自所捕获的细胞或组织的RNA，孵育在RNA裂解缓冲液中的LCM盖膜30分钟，在37℃。 注：对于HS帽，一个适配器可允许用于在盖的使用仅仅10微升裂解缓冲液和附加到0.5 ml离心管。宏帽要求50微升裂解缓冲液，并直接连接到0.5 ml离心管。 </li><li>温育后，旋裂解缓冲液分解成0.5 ml离心管。 </li><li>为了确保所有细胞Ø完全裂解ř组织，剥离瓶盖，发生在裂解液的LCM盖膜。 </li><li>为了测试RNA的完整性，用RNA分离出剩余的后LCM测量RNA完整性幻灯片的组织。 注：由于与LCM获得的有限的样本的RNA，通常不实际的上LCM分离的RNA测试RNA的完整性，但是，从剩余的组织的RNA提供任何RNA降解的良好指标，由于在固定，免疫组织化学和时间LCM过程。 </li></ol>

<ol>
	<li>Emmert-Buck, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection.">Laser capture microdissection.</a> Science. 274, 998-1001 (1996).</li><li>Schutze, K., Lahr, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+expressed+genes+by+laser-mediated+manipulation+of+single+cells.">Identification of expressed genes by laser-mediated manipulation of single cells.</a> Nature biotechnology. 16, 737-742 (1998).</li><li>Schutze, K., Posl, H., Lahr, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+micromanipulation+systems+as+universal+tools+in+cellular+and+molecular+biology+and+in+medicine.">Laser micromanipulation systems as universal tools in cellular and molecular biology and in medicine.</a> Cellular and Molecular Biology. 44, 735-746 (1998).</li><li>Vandewoestyne, M., Van Nieuwerburgh, F., Van Hoofstat, D., Deforce, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+three+DNA+extraction+protocols+for+forensic+STR+typing+after+laser+capture+microdissection.">Evaluation of three DNA extraction protocols for forensic STR typing after laser capture microdissection.</a> Forensic Science International Genetics. 6, 258-262 (2012).</li><li>Eells, J. B., Wilcots, J., Sisk, S., Guo-Ross, S. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NR4A+gene+expression+is+dynamically+regulated+in+the+ventral+tegmental+area+dopamine+neurons+and+is+related+to+expression+of+dopamine+neurotransmission+genes.">NR4A gene expression is dynamically regulated in the ventral tegmental area dopamine neurons and is related to expression of dopamine neurotransmission genes.</a> Journal of Molecular Neuroscience. 46, 545-553 (2012).</li><li>Seelan, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+analysis+of+laser+capture+microdissected+fetal+epithelia.">Epigenetic analysis of laser capture microdissected fetal epithelia.</a> Analytical Biochemistry. 442, 68-74 (2013).</li><li>Kim, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-126+contributes+to+Parkinson's+disease+by+dysregulating+the+insulin-like+growth+factor/phosphoinositide+3-kinase+signaling.">miR-126 contributes to Parkinson's disease by dysregulating the insulin-like growth factor/phosphoinositide 3-kinase signaling.</a> Neurobiology of Aging. 35, 1712-1721 (2014).</li><li>Bohm, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+antidepressant+treatment+on+gene+expression+profile+in+mouse+brain:+cell+type-specific+transcription+profiling+using+laser+microdissection+and+microarray+analysis.">Effects of antidepressant treatment on gene expression profile in mouse brain: cell type-specific transcription profiling using laser microdissection and microarray analysis.</a> Journal of Neurochemistry. 97, 44-49 (2006).</li><li>Kadkhodaei, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factor+Nurr1+maintains+fiber+integrity+and+nuclear-encoded+mitochondrial+gene+expression+in+dopamine+neurons.">Transcription factor Nurr1 maintains fiber integrity and nuclear-encoded mitochondrial gene expression in dopamine neurons.</a> Proceedings of the National Academy of Sciences of the United States of America. 110, 2360-2365 (2013).</li><li>Miller, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Proteomics+in+Laser+Capture+Microdissected+Sleep+Nuclei+From+Rat+Brain.">Quantitative Proteomics in Laser Capture Microdissected Sleep Nuclei From Rat Brain.</a> Journal of Neurogenetics. , (2014).</li><li>Roberts, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+laser+capture+microdissection+and+protein+microarray+technologies+in+the+molecular+analysis+of+airway+injury+following+pollution+particle+exposure.">Application of laser capture microdissection and protein microarray technologies in the molecular analysis of airway injury following pollution particle exposure.</a> Journal of Toxicology and Environmental Health. A. 67, 851-861 (2004).</li><li>Yuferov, V., Nielsen, D., Butelman, E., Kreek, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+studies+of+psychostimulant-induced+changes+in+gene+expression.">Microarray studies of psychostimulant-induced changes in gene expression.</a> Addiction Biology. 10, 101-118 (2005).</li><li>Brown, A. L., Smith, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+RNA+preservation+for+immunolabeling+and+laser+microdissection.">Improved RNA preservation for immunolabeling and laser microdissection.</a> RNA. 15, 2364-2374 (2009).</li><li>Curran, S., McKay, J. A., McLeod, H. L., Murray, G. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microscopy.">Laser capture microscopy.</a> Molecular Pathology. 53, 64-68 (2000).</li></ol>

The authors declare that they have no competing financial interests.

LCM是一个功能强大的技术为细胞或从组织切片的组织的区域的离散种群上的显微镜载玻片和RNA的，微小RNA，DNA和蛋白质的随后分离的夹层。使用高通量技术，如微阵列，下一代测序的RNA，DNA和蛋白组学的后生分析与分析结合使用LCM正变得越来越普遍，这些技术的灵敏度提高，需要原料的量减少8-12。用LCM，一个较好的解剖分辨率实现为一个样本，并从这些样品的下游措施的认识大大提高。一个警告与LCM是它提供了所关注的细胞，但不一定是信元的纯群体的浓缩试样。的精度的限制意味着将有来自相邻组织部分污染。然而，这种技术确实集中感兴趣的细胞尽量减少与周围组织和细胞相关效应和最大程度地在感兴趣的细胞中的实验效果。 直接与间接免疫组化 由于LCM目前利用主要用于隔离和RNA的测量，保持完好的RNA是一个非常重要的标准。维持RNA的完整性是使用间接免疫组织化学的背后的主要理由，引导组织解剖中去除的必要性，以直接染色的组织可降解的RNA（ 图10）。当实验问题，但是，需要的细胞，如多巴胺神经元的特定人群的隔离，直接荧光免疫是必需的。使用快速免疫组织化学标记的协议可在手术过程中的RNA的降解最小化。短的温育时间是由于使用了高浓度的初级和次级抗的机构，浓度在10-20倍，比什么是必要的以2小时培养的常规免疫较高。然而，如果较长的温育时间是必需的，棕色和Smith使用高盐缓冲液来抑制RNA降解，将获得良好的品质的RNA与长孵育时间（达20小时）13。如果存在的组织的标识和获取访问之间需要一个LCM系统大量的时间这种方法也可使用。 PEN膜，硅烷准备和无涂层玻璃幻灯片-幻灯片的选择 为LCM使用未涂覆的玻璃载片已经报道14。在我们的实验室，硅烷制备幻灯片已经被发现是一个最佳的选择，因为该涂层充分地附着在组织到滑动用于免疫组织化学未经组织的损失，但仍允许用于连接组织的到LCM盖和除去从滑动。无涂层的载玻片有表现出一定的组织损失的免疫程序。如果间接免疫组化的情况下，然而，组织滞留变得不再是一个问题。用适当的脱水，捕获细胞或组织脱硅烷制备载玻片尚未一个问题。每个的各种方法，以在本文所述的分离的神经元或脑区都有优点和缺点。对于个别的多巴胺神经元的隔离，利用硅烷制备载玻片具有更好的光学系统的优点，并且比在PEN膜幻灯片便宜。的缺点是，有时捕获细胞可以是困难的，如果有足够的脱水（见下文）和某些组织可能会留在滑动。的PEN膜的幻灯片的优点是，利用红外激光和紫外激光的组合确保了多巴胺的神经元将被收集。失败来收集从PEN膜幻灯片细胞几乎不会发生的，因为它是较少依赖于脱水比载玻片上。一个<html>附加的优点是紫外激光可以用于更接近于感兴趣的神经元的形状，相比于圆形用于捕获神经元关的玻片与红外激光器。对于组织区域的隔离，PEN膜幻灯片远远优于并证明所增加的成本。这种做法的结果完全除去所有切出在膜组织的，比单独使用IR激光和更厚的组织切片可以收集捕获了大面积的组织的速度要快得多。仅使用IR激光器要求在LCM帽膜完全熔化在整个组织区域。此外，收集不全的组织是典型的，通常需要多次尝试。虽然这需要比从PEN膜滑动分离组织更长，如果可用的组织已经在载玻片或UV激光不可用，这是一个可行的选择，因为大多数的组织可以收集（ 图8J）。 ove_content“&gt; 关键步骤 在100％乙醇温育是最重要的步骤之一。为了从硅烷制备载玻片收集细胞的组织的完全脱水是必需的。因此，不除去任何的水，这与100％的乙醇孵育大多关联，将影响到拾取单元从幻灯片的能力。为了解决这个问题，经常改变的100％的乙醇溶液作为从前面的洗涤步骤可影响脱水的空气或转移吸收的水。另外，分子筛用于吸收任何污染。该LCM系统最好应设在一个小房间里用除湿机，以保持较低的湿度条件。 良好的冰冻切片是正确的LCM关键。部分必须平，在最小化的组织褶皱。折叠在组织会增加的距离LCM盖之间，并防止它从接触吨他组织。然后变得难以膜盖充分熔融到组织上。对于载玻片，典型地部分厚度必须是6和12微米之间。较厚的部分可以被捕获与PEN膜的幻灯片。 LCM提供了技术能力，从显微镜玻片使组织非常精确的解剖和细胞。相比的组织片毛解剖这极大地增加了进一步分析的分辨率。虽然，LCM可以是时间和劳动力密集型的，它不需要大量的培训或专业知识，并配合其他高通量技术时，可以大大增强的生物过程的理解，当离散的细胞或解剖区域被使用。

<html>所有组织由细胞的杂合人口。这是特别相关的脑组织中，包括由不同类型的神经胶质细胞（少突胶质细胞，小胶质细胞和星形胶质细胞）的包围各种形态和/或neurochemically明显的神经元。此外，不同的区域在大脑中，如皮质或脑干核的区域，具有特定的功能。因此，能够将分离的细胞或非常小的解剖学不同的区域的特定人群，当与分析技术（ 即 ，Q-PCR，微阵列，RNA测序，和蛋白质组学）结合时，能显着提高各种生物过程的理解。 LCM是一种技术，提供来隔离非常离散的解剖区域或特定的细胞从组织显微镜载玻片上，提供一个更均匀的来源为各种分子，如RNA，微小RNA，DNA和蛋白质的进一步分析的能力。 吨“&gt;自从首次引入的LCM，几种不同的方法已被用来从组织1-3 microdissect细胞。最早的方法包括在幻灯片上直接附着组织的使用红外（IR）激光器和热塑性膜和非使用紫外（UV）激光切割分离的细胞或组织，并收集到一个管接触的方法。接着，LCM已经成功地用于在多种组织和细胞类型，并显示为与隔离各种分子的用于下游分析兼容包括RNA，微小RNA，DNA和蛋白质，包括酶的活性1,4-7这里LCM是使用一个LCM系统，其使用一切割UV激光和用于切割周围的细胞或组织，并将其附加到一个捕获IR激光器证明LCM帽上。该LCM系统采用两个红外捕获激光熔化的塑料薄膜上的帽上，重视的细胞对塑料薄膜和从T中删除所述感兴趣的细胞或组织他组织具有去除帽（ 图1）。另外，在使用红外激光组合，UV激光可用于切出的组织或从组织固定在载玻片上，用膜，然后通过附加的组织，以使用IR激光（ 图2）的LCM帽分离感兴趣的细胞。这些技术的简要概述图1和2中找到。 关于该技术的几个变型被描述于任一隔离用直接荧光免疫组织化学和一个用于组织的基础上的特定蛋白质的表达的区域的隔离的间接免疫组化制导技术特定的细胞。具体地，示出从黑质和腹侧被盖区和RNA从新鲜，冷冻的脑组织的随后分离的隔离多巴胺神经元的分离。作为RNA的是最不稳定的测量分子的（相对于DNA或蛋白质），STEPS帮助保存RNA的完整性都包含与RNA上的所获得的数量和质量的显示的数据。

<table border="0" cellpadding="0" cellspacing="0" width="597">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:159px;">Name of the Material/Equipment</td>
			<td style="width:191px;">Company</td>
			<td style="width:121px;">Catalog Numer&#160;</td>
			<td style="width:127px;">Comments</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:159px;">Veritas Laser Capture Microdissection System</td>
			<td style="width:191px;">Life Technologies, Grand Island, NY</td>
			<td style="width:121px;">Newer model is now sold</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:159px;">CapSure Macro LCM Caps</td>
			<td style="width:191px;">Life Technologies, Grand Island, NY</td>
			<td>LCM0211</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:159px;">CapSure HS LCM Caps</td>
			<td style="width:191px;">Life Technologies, Grand Island, NY</td>
			<td>LCM0213</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:159px;">PEN Membrane slides</td>
			<td style="width:191px;">Life Technologies, Grand Island, NY</td>
			<td>s4651</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:159px;">Silane prep-slides</td>
			<td style="width:191px;">Sigma-Aldrich, St. Louis, MO</td>
			<td>S4651</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:159px;">95% Ethanol-RNase Free</td>
			<td style="width:191px;">Sigma-Aldrich, St. Louis, MO</td>
			<td>E7148</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:159px;">100% Ethanol-RNase Free</td>
			<td style="width:191px;">Sigma-Aldrich, St. Louis, MO</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:159px;">Rnase Free Triton</td>
			<td style="width:191px;">Sigma-Aldrich, St. Louis, MO</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:159px;">Molecular Sieve</td>
			<td style="width:191px;">EDM Millipore, Billerica, MA</td>
			<td>MX1583D</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:159px;">Anti-Tyrosine hydroxyase antibody</td>
			<td style="width:191px;">EDM Millipore, Billerica, MA</td>
			<td>Ab152</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:159px;">Alexa Fluor 488 goat anti-rabbit IgG</td>
			<td style="width:191px;">Life Technologies, Grand Island, NY</td>
			<td>A-11008</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:159px;">2100 Bioanalyzer</td>
			<td style="width:191px;">Agilent Technologies, Santa Clara, CA</td>
			<td>G2939AA</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:159px;">Stratagene MX 3005P</td>
			<td style="width:191px;">Agilent Technologies, Santa Clara, CA</td>
			<td>401513</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:159px;">Nanodrop 3300 fluorospectrometer</td>
			<td style="width:191px;">Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:159px;">Quant-iT RiboGreen</td>
			<td style="width:191px;">Life Technologies, Grand Island, NY</td>
			<td>R11490</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:159px;">RNaseZap</td>
			<td style="width:191px;">Life Technologies, Grand Island, NY</td>
			<td>AM9780</td>
			<td></td>
		</tr>
	</tbody>
</table>

laser capture microdissection - a demonstration of the isolation of individual dopamine neurons and the entire ventral tegmental area

激光捕获显微切割（LCM）用于分离单个细胞或从组织切片的组织的精确解剖区域的人口集中在显微镜载玻片上。当与免疫组织化学结合，LCM可以用来隔离基于特定蛋白质标记单个细胞类型。在这里，液晶显示模块技术进行说明，收集的多巴胺神经元直接标记酪氨酸羟化酶免疫组织化学和用于容纳使用间接酪氨酸羟化酶免疫组在邻近那些用于LCM一节中的腹侧被盖区的区域中的多巴胺神经元的分离的特定人口。一个红外（IR）激光捕获用于两个解剖单个神经元，以及腹侧被盖区离开玻璃载玻片和到一个LCM盖进行分析。用100％乙醇和二甲苯的组织完全脱水是至关重要的。红外激光捕获和紫外（UV）cutti组合纳克激光被用于使用的PEN膜的幻灯片时，以分离单个的多巴胺神经元或腹侧被盖区。笔膜滑动在载玻片上具有显著优势，因为它提供了更好的一致性在捕获和收集单元，被更快收集大块的组织，是在脱水和结果较少依赖于完全除去从滑动的组织。虽然移除大面积的组织从载玻片是可行的，这是相当耗费更多的时间，并经常留下一些残余组织。这里显示的数据表明，可以使用这些程序的定量PCR测量得到的足够数量和质量的RNA。尽管RNA和DNA是最常见的分离分子从组织和细胞中收集与微小RNA，蛋白质和DNA的表观遗传变化的LCM，分离和测定也可从增强的解剖和蜂窝分辨率有益于使用LCM获得的。

在图6和7的显微照片表明个别多巴胺神经元的分离的功能。当前解剖分辨率为大约5-10微米的作为能够始终如一地产生在LCM的最小斑点尺寸帽以分离细胞。对于特定细胞类型的分离的唯一限制是可视化的细胞的能力。虽然LCM能够隔离单个多巴胺神经元，相邻的未标记细胞的部分污染最可能发生。因此，最后的样品是浓集多巴胺神经元，而不是多巴胺神经元的纯群体。这一技术也能够分离组织的小区域，如大脑内的离散的细胞核或区域如图所示与腹侧被盖区的隔离。以前的出版物已经表明在脑组织中此使用以及从正常组织5,11-隔离病理组织。 <p类=“jove_content”&gt;因为最常见的用途的细胞和组织使用LCM隔离是RNA分析，提出的程序进行了优化，以保持RNA的完整性。为了确定RNA的质量仍然以下LCM，分离RNA，使用基于硅石离心柱，从以下的LCM幻灯片组织，无论之后在丙酮中快速酪氨酸羟化酶荧光免疫组织化学过程或固定。 RNA的质量进行测定，并进行比较，以用于从可商购的全脑的RNA的RNA的值（ 图9）。 RNA的质量减少来自组织的RNA样本免疫后用于LCM具有较低完整性（RIN不可用），然后加入丙酮固定（RIN 2.6）中相比，全脑的RNA（RIN 8.2），为可以通过在一个相对减少可见所述的rRNA S28峰相比，S18峰高度。然而，该RNA保持18S和28S频带和基因表达可以用Q-PCR来测量。 图10A）中分离。以测量的RNA量与Q-PCR，一系列的全脑的RNA和RNA从多巴胺神经元的浓度逆转录和β肌动蛋白基因使用Q-PCR如前所述5进行测定。基于这些测量，3.55，6.82和20.58微克/微升的浓度是从50，100和200的多巴胺神经元，分别（ 图10B）来计算。 来演示如何多巴胺神经元的隔离比较整个诉隔离与LCM entral被盖区，多巴胺神经元特异性基因（Nurr1的，酪氨酸羟化酶和多巴胺转运体），测定用Q-PCR在这两个不同类型的样品和比较β肌动蛋白（ 图11）的表达。因为是与感兴趣的基因的较高浓度下产生的（C T）的值，在ΔC 吨减少表示相对于β肌动蛋白的多巴胺神经元的基因表达增加。这说明了如何从个人的多巴胺神经元的分离浓缩物中的多巴胺神经元特异性基因的表达。在腹侧被盖区的样品，多巴胺神经元特异性基因稀释了作为其他细胞（非多巴胺能神经元和神经胶质细胞）也将被收集表达β肌动蛋白，但不表达的多巴胺神经元的基因。 <img alt="图1" src="/files/ftp_upload/52336/52336fig1highres.jpg" /> <stro纳克&gt;使用红外线（IR）激光捕获图1.激光捕获的小塑料LCM盖具有上被放置在组织安装到载玻片（步骤1）的顶部的底部的膜。当组织或感兴趣的细胞被识别，一个IR捕获激光通过LCM帽烧成熔化的小凹坑在膜到下面的组织/细胞（步骤2）。当LCM盖被移除，所述组织是从显微镜载玻片取出并保持附着到LCM帽。 <img alt="图2" src="/files/ftp_upload/52336/52336fig2highres.jpg" /> 图2.激光捕获使用红外线（IR）激光捕获和紫外（UV）激光切割，为了获得更大的组织区域或便于使用紫外激光的细胞的回收，组织被安装在与膜滑动连接沿着弯道的外侧（PEN膜幻灯片）的幻灯片。 LCM的帽放置在吨他组织和IR捕获激光被用来熔化帽膜和附加组织到LCM盖（步骤1和2）。紫外激光切割，然后用通过滑动膜和组织（步骤3和4），这样，当帽被去除的组织被从幻灯片除去残留在LCM帽（步骤5）来切断。 <img alt="图3" src="/files/ftp_upload/52336/52336fig3highres.jpg" /> 图3.激光捕获显微切割系统的路线图。这激光捕获显微切割系统具有3张幻灯片的时间空间。当载玻片装入显微镜中，产生低倍率路线图的图像对每个幻灯片。路线图图像上选择一个区域移动实时图像窗口的区域。出于演示的目的，鼠标中脑被划分为10微米节到3张幻灯片。第一滑动被标记为酪氨酸羟化酶immunoreactivi用二氨基联苯胺作为发色（A）TY。部分被安装在任一硅烷制备载玻片（B）或PEN膜幻灯片（℃）。这两个幻灯片采用了快速荧光酪氨酸羟化酶免疫组化标记。 （C）中箭头表示的PEN膜的玻璃载片上的附着。该境外任何组织可以收集与LCM。 <img alt="图4" src="/files/ftp_upload/52336/52336fig4highres.jpg" /> 图4.使用红外激光捕获的捕获红外激光被用来熔化LCM帽膜到组织附着到LCM盖和从组织的其余部分中取出。红外激光捕获必须以足够的力量来融化LCM盖膜充分接触下层组织和搏命。上面的图片展示了当激光不足以熔化膜到所述滑动件（左两个点）。当融化膜接触载玻片，厚厚的黑色轮廓可以看出（右点）。激光的强度可以通过改变功率（3-100毫瓦），脉冲（100-1,000,000微秒）和＃命中激光器进行调整。另外，反复的IR激光器的烧成在同一地点也可以进一步熔融膜。需要红外激光的目标进行调整，随时LCM盖被移动或当物镜被改变。比例尺= 100微米。 <img alt="图5" src="/files/ftp_upload/52336/52336fig5highres.jpg" /> 图5.使用紫外激光切割，紫外激光切割用于通过PEN膜和组织切割。在使用前需要削减通过膜和组织需要被确定，因为UV激光可损害组织的最小强度。以上的三个点不同的紫外激光强度（箭头）显示与<html>左光点尺寸足够10微米的部分，中间点为较厚部分和右侧的点表明UV激光强度太高。比例尺= 100微米。 <a href="https://www.jove.com/files/ftp_upload/52336/52336fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/52336/52336fig6highres.jpg" /> 图使用红外激光酪氨酸羟化酶阳性神经元6.直接隔离。多巴胺神经元的酪氨酸羟化酶（A）快速荧光标记后显示。当IR激光器进行烧成熔化 ​​在这些神经元（D）中的LCM帽膜，除去盖的拾取这些细胞离滑动件（B，E）的。这些神经元可以被可视化为附着到LCM盖（C，F）。比例尺= 250微米。=“htt​​ps://www.jove.com/files/ftp_upload/52336/52336fig6large.jpg”目标=“_空白”&gt;点击此处查看该图的放大版本。 <img alt="图7" src="/files/ftp_upload/52336/52336fig7highres.jpg" /> 图从PEN膜幻灯片使用红外和紫外激光器酪氨酸羟化酶阳性神经元7.直接隔离。酪氨酸羟化酶标记神经元以上（A）所示。这些神经元可被附连到使用IR激光的LCM盖和切断从使用紫外激光（B）的在PEN膜滑动。比例尺= 250微米。 <a href="https://www.jove.com/files/ftp_upload/52336/52336fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图8" src="/files/ftp_upload/52336/52336fig8highres.jpg" /> 图8.间接<html>使用酪氨酸羟化酶免疫反应性的腹侧被盖区的隔离。在腹侧被盖区的多巴胺神经元的位置使用标准免疫组织化学技术（A和E）中的可视化中的一节。此免疫标记的部分被用作模板来定位腹侧被盖区中的未染色相邻部分（B和F）。使用UV激光器，腹侧被盖区附着到LCM帽与红外激光器和切断从与紫外激光（C和D）滑动并且被示出附连到的HS LCM盖（D）中 。腹侧被盖区，从硅烷制备载玻片仅使用红外激光分离还示出连接到一个宏帽（FK）。注意，一个以上的尝试，需要对收集大部分的组织从该区域（比较G和 J）。比例尺= 500微米。PLOAD / 52336 / 52336fig8large.jpg“目标=”_空白“&gt;点击此处查看该图的放大版本。 <img alt="图9" src="/files/ftp_upload/52336/52336fig9highres.jpg" /> 图9. RNA的质量。代表电泳和市售全脑的RNA（A）和RNA的分离部分丙酮固定和LCM（B）的后或快速荧光酪氨酸羟化酶免疫组织化学及LCM（C）的相关联的凝胶图像。虽然RNA质量降低处理用于免疫组织化学，比起全脑的RNA区段，所述电泳表明大部分完整的RNA基于所述rRNA的S18和S28中的峰的存在。全脑RNA浓度为6.487微克/微升8.2一个RIN。丙酮固定组织RNA浓度为5.036微克/微升用2.6 RIN。免疫组化后ħRNA获得<html>广告4.474皮克/微升和RIN浓度不可用。 <img alt="图10" src="/files/ftp_upload/52336/52336fig10highres.jpg" /> 图10.核糖核酸数量。为了确定在与LCM获得的神经元的RNA的量，RNA浓度的标准曲线是使用fluorospectrometer和Q-PCR技术制备。为fluorospectrometer测量（A）中，较大的曲线图示出了较高范围的RNA量和小图显示了该测定的灵敏度下降到10微克/微升。从50，100和200的多巴胺神经元获得的RNA的浓度为17.3，24.8和50.9微克/微升，分别。使用Q-PCR来测量RNA的量（B）中 ，RNA的浓度为50，100和200的多巴胺神经元得到的是3.55，6.82和20.58微克/微升，分别。 ES / ftp_upload / 52336 / 52336fig11highres.jpg“/&gt; 的多巴胺神经元特异性基因的个体的多巴胺神经元的分离图11浓度的影响。在（C T）（ΔCT）的多巴胺神经元特异性基因（Nurr1的，酪氨酸羟化酶和多巴胺转运体）和β-actin之间在多巴胺神经元的样品的差在腹侧被盖区分离的LCM相比在整个腹侧被盖区的解剖表达被示出。因为是与感兴趣的基因的较高浓度下产生的（C T）的值，在ΔC 吨减少表示相对于β肌动蛋白作为其他细胞（非多巴胺能神经元和神经胶质细胞）的多巴胺神经元的基因表达的增加将收集表达β肌动蛋白，但不表达的多巴胺神经元的基因的腹侧被盖区中的样品。

Watch this Scientific Journal Video about Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area at JoVE.com

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

个别的多巴胺神经元的分离或用直接或间接的免疫组织化学的腹侧被盖区，使用激光捕获显微切割证实。对于使用红外激光从玻璃载片隔离的组织，并从用红外和紫外激光的组合膜载玻片参数进行了讨论。

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 ...

laser-capture-microdissection-demonstration-isolation-individual

Research

JoVE Journal

Neuroscience

23.8K 次观看.  Mississippi State University College of Veterinary Medicine. 个别的多巴胺神经元的分离或用直接或间接的免疫组织化学的腹侧被盖区，使用激光捕获显微切割证实。对于使用红外激光从玻璃载片隔离的组织，并从用红外和紫外激光的组合膜载玻片参数进行了讨论。 

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

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 ...

科研

神经科学

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

总RNA的分离和下游下一代测序和基因表达谱的非激光捕获显微切割离散小鼠脑地区显微镜方法

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five decades of inquiry into the molecular deficiencies associated with dopamine regulation. The majority of these inquiries of dopamine regulation in the brain focus upon the molecular basis for its regulation in the terminal field regions of the nigrostriatal and mesoaccumbens pathways; striatum and nucleus accumbens. Furthermore, such studies have concentrated on analysis of dopamine tissue content with normalization to only wet tissue weight. Investigation of the proteins that regulate dopamine, such as tyrosine hydroxylase (TH) protein, TH phosphorylation, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) protein often do not include analysis of dopamine tissue content in the same sample. The ability to analyze both dopamine tissue content and its regulating proteins (including post-translational modifications) not only gives inherent power to interpreting the relationship of dopamine with the protein level and function of TH, DAT, or VMAT2, but also extends sample economy. This translates into less cost, and yet produces insights into the molecular regulation of dopamine in virtually any paradigm of the investigators' choice.
We focus the analyses in the midbrain. Although the SN and VTA are typically neglected in most studies of dopamine regulation, these nuclei are easily dissected with practice. A comprehensive readout of dopamine tissue content and TH, DAT, or VMAT2 can be conducted. There is burgeoning literature on the impact of dopamine function in the SN and VTA on behavior, and the impingements of exogenous substances or disease processes therein 1-5. Furthermore, compounds such as growth factors have a profound effect on dopamine and dopamine-regulating proteins, to a comparatively greater extent in the SN or VTA 6-8. Therefore, this methodology is presented for reference to laboratories that want to extend their inquiries on how specific treatments modulate behaviour and dopamine regulation. Here, a multi-step method is presented for the analyses of dopamine tissue content, the protein levels of TH, DAT, or VMAT2, and TH phosphorylation from the substantia nigra and VTA from rodent midbrain. The analysis of TH phosphorylation can yield significant insights into not only how TH activity is regulated, but also the signaling cascades affected in the somatodendritic nuclei in a given paradigm.
We will illustrate the dissection technique to segregate these two nuclei and the sample processing of dissected tissue that produces a profile revealing molecular mechanisms of dopamine regulation in vivo, specific for each nuclei (Figure 1).

Dopamine is distinctly regulated in the midbrain nuclei, which contain the cell bodies and dendrites of the dopamine neurons. Here we describe a dissection and sample-handling approach to maximize results, and thus conclusions and insights, on dopamine regulation in the midbrain nuclei of the substantia nigra (SN) and ventral tegmental area (VTA) in rodents.

在黑质和腹侧被盖区多巴胺调控的综合性能分析

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

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 ...

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments.&#160;In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent &#60;10% of the total cell population.&#160;Laser capture microdissection (LMD) has been developed to address this problem.&#160;Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited.&#160;LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity.&#160;Standard techniques are used to recover the total RNA and measure its integrity.&#160;This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

High-quality total RNA has been prepared from cell bodies of mouse spinal cord motor neurons by laser capture microdissection after staining spinal cord sections with Azure B in 70% ethanol.&#160;Sufficient RNA (~40-60 ng) is recovered from 3,000-4,000 motor neurons to allow downstream RNA analysis by RNA-seq and qRT-PCR.

通过激光捕获微分路，从小鼠脊髓运动神经细胞体中生成RNA进行转录分析

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

A Method to Make a Craniotomy on the Ventral Skull of Neonate Rodents

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain in adulthood and during development. Although most in vivo approaches use a craniotomy to study brain regions located on the dorsal side, brainstem regions such as the pons, located on the ventral side remain relatively understudied. The main goal of this protocol is to facilitate access to ventral brainstem structures so that they can be studied in vivo using electrophysiological and imaging methods. This approach allows study of structural changes in long-range axons, patterns of electrical activity in single and ensembles of cells, and changes in blood brain barrier permeability in neonate animals. Although this protocol has been used mostly to study the auditory brainstem in neonate rats, it can easily be adapted for studies in other rodent species such as neonate mice, adult rodents and other brainstem regions.

A surgical method is described to expose the ventral skull in neonate rats. Using this approach it is possible to open a craniotomy to perform acute electrophysiology and two-photon microscopy experiments in the brainstem of anesthetized pups.

方法以使对新生儿鼠害的腹头骨开颅手术

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain ...

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium carbonate. Animal studies as well as human genetic studies indicate that lithium affects molecular targets that are involved in neuronal growth, survival and maturation, and notably molecules involved in Wnt signaling. Given the ethical challenge to obtaining brain biopsies for investigating dynamic molecular changes associated with lithium-response in the central nervous system (CNS), one may consider the use of neurons obtained from olfactory tissues to achieve this goal.The olfactory epithelium contains olfactory receptor neurons at different stages of development and glial-like supporting cells. This provides a unique opportunity to study dynamic changes in the CNS of patients with neuropsychiatric diseases, using olfactory tissue safely obtained from nasal biopsies. To overcome the drawback posed by substantial contamination of biopsied olfactory tissue with non-neuronal cells, a novel approach to obtain enriched neuronal cell populations was developed by combining nasal biopsies with laser-capture microdissection. In this study, a system for investigating treatment-associated dynamic molecular changes in neuronal tissue was developed and validated, using a small pilot sample of BD patients recruited for the study of the molecular mechanisms of lithium treatment response.

In this study, a novel platform to investigate intraneuronal molecular signatures of treatment response in bipolar disorder (BD) was developed and validated. Olfactory epithelium from BD patients was obtained through nasal biopsies. Then laser-capture microdissection was combined with Real Time RT PCR to investigate the molecular signature of lithium response in BD.

通过鼻腔活检获得联合激光捕获显微切割的嗅觉神经元：一种可能的方法来研究治疗反应的精神障碍

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium ...

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, interactions with our environment can induce brain plasticity. Increasingly, research is trying to identify which environments stimulate brain plasticity beneficial for treating brain and behavioral disorders. Two environmental manipulations are described which increase or decrease the number of tyrosine hydroxylase immunopositive (TH+, the rate-limiting enzyme in dopamine (DA) synthesis) neurons in the adult mouse midbrain. The first comprises pairing male and female mice together continuously for 1 week, which increases midbrain TH+ neurons by approximately 12% in males, but decreases midbrain TH+ neurons by approximately 12% in females. The second comprises housing mice continuously for 2 weeks in &#8216;enriched environments&#8217; (EE) containing running wheels, toys, ropes, nesting material, etc., which increases midbrain TH+ neurons by approximately 14% in males. Additionally, a protocol is described for concurrently infusing drugs directly into the midbrain during these environmental manipulations to help identify mechanisms underlying environmentally-induced brain plasticity. For example, EE-induction of more midbrain TH+ neurons is abolished by concurrent blockade of synaptic input onto midbrain neurons. Together, these data indicate that information about the environment is relayed via synaptic input to midbrain neurons to switch on or off expression of &#8216;DA&#8217; genes. Thus, appropriate environmental stimulation, or drug targeting of the underlying mechanisms, might be helpful for treating brain and behavioral disorders associated with imbalances in midbrain DA (e.g. Parkinson&#8217;s disease, attention deficit and hyperactivity disorder, schizophrenia, and drug addiction).

This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

中脑多巴胺神经元的数目在成年小鼠环境的调变

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.