我们要感谢Skok实验室，特别是苏珊娜·休伊特，成员的讨论和意见。这项工作是由美国国家卫生补助R01GM086852，RC1CA145746（JAS）的支持。 JAS是一个白血病和淋巴瘤协会的学者。 JC是欧文顿研究所的癌症研究所的研究员。 MM是由美国国家科学基金会资助中西医结合研究生教育和研究培训实习（IGERT 0333389 NSF）的支持。

1。 DNA探针标记荧光基团：：尼克翻译（约6小时） <ol><li>清洁的BAC DNA（其制备由马克西制备）或质粒或PCR产物，所有的再悬浮在H 2 O中，可用于标记， 请注意 ，一个强大的FISH信号，探针应跨越至少10 kb的。 </li><li>孵育DNA的RNA酶A中的30分钟，在37℃（ 表1中列出的所有试剂）。 </li><li>在16°C孵育2小时缺口翻译反应（见表2和表3）。直接标记的替代方法，可以使用（实施例：FISH标签，Invitrogen公司）。 </li><li>失活反应1小时，在-80℃或-20℃下过夜</li><li>确定探头的尺寸，在2％琼脂糖凝胶上。涂片应介于100和1000bp的，与多数在约300-500 bp的（ 请注意 ，在涂抹的Cy5探针是不可见的）。如果是不能消化足够的DNA，DNA聚合酶I和DNA酶I可以加入到该反应，并在16℃下温育两小时</li><li>在两升的烧杯中，充满了H 2 0 2小时在黑暗中的0.025毫米过滤器净化探针。 </li><li>添加阻断因素的探针如下：婴儿床-1的DNA的10微克，10微克Hybloc DNA，鲑鱼精子DNA探针的10微克和100微克。 DNA探针存储于-20℃下</li></ol> 2。探头沉淀/变性/预退火（3-4小时） <ol><li>之间的0.3微克，1微克的DNA探针是用于每盖玻片。用0.1倍体积的3M乙酸钠（pH 5.2）和2.5倍体积的100％乙醇的混合探针，温育1小时，在-80℃或在-20℃下过夜，降速以13,000 rpm在4℃下30分钟。 请注意 ，根据质量的DNA和杂交的核区域，DNA探针的量可被调整。直接标记的替代方法，可以允许使用的减少量的探头。有关一个幻灯片的多个DNA FISH信号的检测，不同的探针可以一起沉淀（除了这不应该是预退火后，类似重复序列）的探针。也可以在同一时间进行的几个实验的探针一起沉淀。浅色的颗粒应根据荧光（如果少量的探针沉淀，沉淀可能是不可见的）。 </li><li>干燥颗粒，并重新悬浮于15μl杂交缓冲液（ 见表4）每盖玻片振荡（使用的Eppendorf恒温）在黑暗中于37-45℃（约20分钟）。 </li><li>为5分钟，在75-95℃下的变性探针</li><li>预退火的探针在37℃下30-60分钟。可调节长度的预退火，根据探针的复杂性和质量。 </li><li>将探针杂交过夜的盖玻片（见第3节）。 </li></ol> 3。 3二维DNA FISH - 第一天（约3小时） <ol><li>非粘附细胞在小体积的1x PBS（〜2×10 5个细胞在5-10微升1×PBS中，每盖玻片）浓缩和丢弃的中心上的聚-L-赖氨酸涂布的盖玻片。应培养贴壁细胞的盖玻片上，在至少24小时（〜70％汇合）（盖玻片可涂覆有0.1％明胶）。广场18x18的24-24毫米的盖玻片放置在6孔培养板中，并在整个协议，两个毫升的各溶液中，每孔使用（可以放置在12/24-well板交替圆1.5毫米盖玻片）。 </li><li>修复细胞在2％多聚甲醛/ 1×PBS，pH值为7-7.4，在RT（4％PFA股票可以等分，储存在-20℃下）10分钟。在溶液中可以购买多聚甲醛（16％），或4％的股份解决方案，可制取粉末/小球。多聚甲醛的粉末溶解在1x PBS，在&gt; 60°C（可提高pH值至8-9，以促进溶解），在冰上的液冷式，调整pH至7-7.4，过滤，分装在隼周贮存于-20℃下</li><li>三个在1x PBS漂洗后，透性细胞在冰冷的0.4％的Triton-X-100/1×PBS中，在冰上5分钟。调整长度的通透性，根据细胞类型。 注意 ：，DNA FISH与免疫相结合，应进行免疫组化，在这个阶段（见第4章 ）。 </li><li>三个在1x PBS漂洗，孵育细胞后，与0.1微克/微升的核糖核酸酶A在1x PBS，1小时，在37℃下盖玻片置于细胞的一面朝下下降了100μl的解决方案封口膜在潮湿的室内或幻灯片上。盖玻片，然后小心地用钳子取出。如果任何遇到阻力，盖玻片应被淹没用1×PBS，以避免损坏细胞。 </li><li>三个在1x PBS漂洗后，在冰冷的0.7％的Triton-X-100 / 0.1 M盐酸透性细胞在冰上10分钟。 </li><li>经过三次冲洗我Ň1X PBS，在1.9 M HCl 30分钟，在室温下变性细胞。或者，细胞可以在50％甲酰胺/ 2×SSC中变性，在75-80℃下至少30分钟。 请注意 ，杂交的长度（和温度）取决于细胞类型可被优化。 </li><li>三个在冰冷的1×PBS中漂洗后，在一个黑暗，潮湿的腔室中，在37℃下过夜，与探针杂交的细胞。盖玻片放置单元侧下降到滴15μl的探针在幻灯片上，并密封与橡胶水泥。 </li></ol> 4。 3维免疫FISH - 第一天（约6-7小时） <ol><li>结合免疫荧光/ DNA的FISH，免疫染色后应进行0.4％的Triton-X-100（ 步骤3.3）中的通透性。 </li><li>孵育细胞30分钟，在RT（2.5％牛血清白蛋白（BSA），10％正常山羊血清，0.1％Tween-20的）的封闭溶液。阻挡的溶液过滤，等分在1.5ml Eppendorfs和存储为months于-20℃下</li><li>孵育细胞与第一抗体反对封闭溶液稀释的感兴趣的蛋白或组蛋白修饰，在潮湿的腔室在RT 1小时。在这里，我们将展示的例子中的抗体对磷酸化丝氨酸139 H2AX（γ-H2AX，Millipore公司的详细信息，请参阅表5）。盖玻片置于细胞的一面朝下下降了100μl的解决方案封口膜在潮湿的室内或幻灯片上。盖玻片然后小心地用钳子（充斥如有必要，用1×PBS）除去注意的抗体和细胞类型的质量取决于稀释和孵化长度可以调整。 </li><li>洗涤细胞，在0.2％BSA / 0.1％的Tween-20/1×3倍的PBS中，在RT下振摇5分钟。 </li><li>孵育细胞与稀释在封闭溶液在RT 1小时在一个黑暗，潮湿的腔室中的相应的荧光团共轭的次级抗体。这里，我们表明检测的一个例子与次要的羊抗鼠抗体（的Alexa Fluor 488或555，Invitrogen公司的详细信息，请参阅表5）。半抗原缀合的第二抗体（生物素，地高辛等），也可以使用。在这种情况下，一个额外的步骤，用于适当的检测（链霉抗生物素蛋白，抗挖，等）可以被执行之前或之后，过晚DNA-FISH杂交。 </li><li>漂洗细胞在0.1％的Tween-20/1×PBS中，3次5分钟的在RT振荡在黑暗中。 </li><li>修复后的细胞在2％多聚甲醛/ PBS洗涤10分钟，在室温下，在黑暗中。 </li><li>继续DNA FISH以上核糖核酸酶A（3.4）从孵化。 </li></ol> 5。 3维DNA FISH /免疫FISH - 第二日（约2小时） <ol><li>小心地取出橡皮泥，洪水盖玻片用2×SSC，用钳子小心地取出，并将其放置在洗涤液孔中。 </li><li>洗涤细胞2倍SSC，30分钟，37°C振荡在黑暗中。</li><li>洗涤细胞2倍SSC在室温为30分钟，在黑暗中晃动。 </li><li>在1x SSC洗涤细胞，在黑暗下振荡30分钟，在RT。 请注意，在用甲酰胺变性的情况下，清洗应该由下列替代：洗涤3次，在50％甲酰胺/ 2×SSC洗涤3次，在2×SSC，5最小，在37°C。 </li><li>挂载的ProLong黄金安装介质，含有1.5微克/毫升的DAPI（4,6 - 二脒基-2 - 苯基吲哚）对染的总DNA的细胞。下降10-15μl的安装介质上的幻灯片上，盖玻片放在细胞和密封的指甲油。幻灯片数月保持在-20°C </li></ol> 6。的3D显微镜和分析<ol><li>不同的显微镜系统，三维图像可以被收购。在我们的实验中，0.3微米的光学部分分离，收集上的Leica SP5 AOBS的共聚焦系统（声光分束器） 之间的分离光学升飞机可以调整取决于分析的类型。 </li><li> 3D图像栈可以使用不同的软件和工具进行分析。我们使用Image J软件进行评估利益的位点之间的距离测量和分析不同类型的利益与核车厢或蛋白质的基因位点的关联。 </li><li>进行所有的统计分析来比较两个（或更多）不同的生物与成对评估（s）的意义：不同的细胞类型或阶段之间的比较，比较的利益的不同基因座之间的条件。在所有的统计检验，P值≤5.00E-2（α= 0.05），是显着的（1.00E-2≤P≤5.00E-2 *显著; 1.00E-3≤P≤1.00E-2 * *非常显著，P &lt;1.00E-3 ***非常显着）。 </li></ol> 两个等位基因之间的距离在整个分布的统计分析，我们第一个c累积分布频率曲线onstruct的整个范围内的测量之间的距离的两个alleles.To评估我们使用非参数两样本Kolmogorov-Smirnov测试（KS）13,14的实证等位基因间的距离的分布中的差异的意义。 统计分析的两个等位基因的最佳距离截止密切的联系（即配对）。要确定范围内的最强大的两个等位基因位点之间的距离差异，我们采用了一系列的双尾Fisher精确检验15秒 。也就是说，在每个测量距离我们测试一个条件是否显着超额代表在较短的两个样本之间的距离。的最小的 P值的分布中表示的范围内的距离，应被看作是密切关联的两个等位基因（ 即配对）的截止。随后，双尾Fisher精确检验采用tØ评估在识别cut-off值15的配对两个等位基因的意义。多次测试修正被应用到占总数的费舍尔测试的执行16。 利益与核车厢或蛋白质的轨迹协会的统计分析。，双尾费舍尔精确的测试是用来分析的意义协会感兴趣的轨迹与不同的车厢或蛋白质15。

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pre-B-cell+receptor+induces+silencing+of+VpreB+and+lambda5+transcription.">The pre-B-cell receptor induces silencing of VpreB and lambda5 transcription.</a> Embo J. 24, 3895-3905 (2005).</li><li>Roldan, 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=Locus+'decontraction'+and+centromeric+recruitment+contribute+to+allelic+exclusion+of+the+immunoglobulin+heavy-chain+gene.">Locus 'decontraction' and centromeric recruitment contribute to allelic exclusion of the immunoglobulin heavy-chain gene.</a> Nature immunology. 6, 31-41 (2005).</li><li>Skok, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonequivalent+nuclear+location+of+immunoglobulin+alleles+in+B+lymphocytes.">Nonequivalent nuclear location of immunoglobulin alleles in B lymphocytes.</a> Nature. 2, 848-854 (2001).</li><li>Skok, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+contraction+by+looping+of+the+Tcra+and+Tcrb+loci+in+rearranging+thymocytes.">Reversible contraction by looping of the Tcra and Tcrb loci in rearranging thymocytes.</a> Nature immunology. 8, 378-387 (2007).</li><li>Xiang, Y., Zhou, X., Hewitt, S. L., Skok, J. A., Garrard, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multifunctional+element+in+the+mouse+Igkappa+locus+that+specifies+repertoire+and+Ig+loci+subnuclear+location.">A multifunctional element in the mouse Igkappa locus that specifies repertoire and Ig loci subnuclear location.</a> Journal of Immunology. 186, 5356-5366 (2011).</li><li>Hewitt, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RAG-1+and+ATM+coordinate+monoallelic+recombination+and+nuclear+positioning+of+immunoglobulin+loci.">RAG-1 and ATM coordinate monoallelic recombination and nuclear positioning of immunoglobulin loci.</a> Nature immunology. 10, 655-664 (2009).</li><li>Deriano, L., 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=The+RAG2+C+terminus+suppresses+genomic+instability+and+lymphomagenesis.">The RAG2 C terminus suppresses genomic instability and lymphomagenesis.</a> Nature. 471, 119-123 (2011).</li><li>Brown, K. E., Baxter, J., Graf, D., Merkenschlager, M., Fisher, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+repositioning+of+genes+in+the+nucleus+of+lymphocytes+preparing+for+cell+division.">Dynamic repositioning of genes in the nucleus of lymphocytes preparing for cell division.</a> Molecular cell. 3, 207-217 (1999).</li><li>Fernandez-Capetillo, O., Lee, A., Nussenzweig, M., Nussenzweig, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H2AX:+the+histone+guardian+of+the+genome.">H2AX: the histone guardian of the genome.</a> DNA repair. 3, 959-967 (2004).</li><li>Croft, J. 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=Differences+in+the+localization+and+morphology+of+chromosomes+in+the+human+nucleus.">Differences in the localization and morphology of chromosomes in the human nucleus.</a> The Journal of cell biology. 145, 1119-1131 (1999).</li><li>Chaumeil, J., Le Baccon, P., Wutz, A., Heard, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+role+for+Xist+RNA+in+the+formation+of+a+repressive+nuclear+compartment+into+which+genes+are+recruited+when+silenced.">A novel role for Xist RNA in the formation of a repressive nuclear compartment into which genes are recruited when silenced.</a> Genes Dev. 20, 2223-2237 (2006).</li><li>Walter, J., 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=Towards+many+colors+in+FISH+on+3D-preserved+interphase+nuclei.">Towards many colors in FISH on 3D-preserved interphase nuclei.</a> Cytogenetic and genome research. 114, 367-378 (2006).</li><li>Toomre, D., Bewersdorf, 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+new+wave+of+cellular+imaging.">A new wave of cellular imaging.</a> Annual review of cell and developmental biology. 26, 285-314 (2010).</li><li>Schermelleh, L., Heintzmann, R., Leonhardt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+super-resolution+fluorescence+microscopy.">A guide to super-resolution fluorescence microscopy.</a> The Journal of cell biology. 190, 165-175 (2010).</li><li>Dobbie, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OMX:+a+new+platform+for+multimodal,+multichannel+wide-field+imaging.">OMX: a new platform for multimodal, multichannel wide-field imaging.</a> Cold Spring Harbor protocols. , 899-909 (2011).</li><li>Boyle, S., Rodesch, M. J., Halvensleben, H. A., Jeddeloh, J. A., Bickmore, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+situ+hybridization+with+high-complexity+repeat-free+oligonucleotide+probes+generated+by+massively+parallel+synthesis.">Fluorescence in situ hybridization with high-complexity repeat-free oligonucleotide probes generated by massively parallel synthesis.</a> Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology. 19, 901-909 (2011).</li></ol>

作者宣称，他们没有竞争的金融利益。

上面详细介绍了在我们的实验室中使用的技术分析的调节V（D）J重组的免疫球蛋白的TCRA / D位点在发展中淋巴细胞30,31。我们相信，这种技术可以适用于不同的核蛋白质，核车厢及基因位点的检测，在不同的细胞类型。协议的修改可能是必要的，而且在这种情况下的主要步骤，集中有以下几种。首先，可以调整长度通透性取决于细胞类型。二初级抗体孵育的长度也可以被调整?...

表观遗传机制的发育和细胞类型特异的转录图谱的的触发建立和继承。在一个层面上，这涉及到调制的定义激活或沉默的基因组区域的染色质包装。在一个规模较大的基因组和核架构，全球3D组织也发挥了作用，在控制转录模式。因此，解剖这些表观基因的功能是必不可少的一个全面的了解基因是如何调控6-11。 结合免疫荧光和三维DNA鱼类提供了一个独特的机会，以补充分子生物学和生物化学的分析评估特定的DNA序列和/或细胞核内的蛋白质的相互作用/协会。此外，在全基因组高通量技术，如染色质免疫沉淀（ChIP-seq的）或染色体捕捉构象的深度测序（4C-seq技术，5C，加上HI-C）提供全球性的DAT一的细胞群12，免疫荧光/ DNA荧光原位杂交技术在单细胞水平上能够进行分析。 在这里，我们描述了一个协议，用于同时检测组蛋白修饰免疫荧光法和DNA序列的DNA荧光原位杂交，然后通过的3D显微镜和分析（3D免疫FISH）。此协议的优点是DNA和保存的蛋白质结构的组合的可视化。我们在这一领域的经验，使我们能够改进和简化现有的协议。虽然我们已经使用该协议来检测在淋巴细胞发生重组的DNA双链断裂，该方法可以应用到其他的蛋白质和其他细胞类型。

<table border="1"><tbody><tr><td> 的试剂/材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> H 2 O </td><td>费舍尔</td><td> ＃BP2470 </td><td></td></tr><tr><td>核糖核酸酶A </td><td>西格玛</td><td> ＃R4642 </td><td></td></tr><tr><td> DNTP </td><td>西格玛</td><td> ＃DNTP100 </td><td></td></tr><tr><td> Alexa的原位</td><td> Invitrogen公司</td><td> ＃C11397 C-11401 </td><td></td></tr><tr><td> Cy3或Cy5的dUTP </td><td>费舍尔</td><td> ＃45-001-XXX </td><td></td></tr><tr><td> DNA酶I </td><td>罗氏公司</td><td> ＃04536282001 </td><td></td></tr><tr><td> DNA聚合酶I </td><td>生物实验室</td><td> ＃M0209 </td><td></td></tr><tr><td> 0.025微米的过滤器</td><td> MilliporË </td><td> ＃VSWP02500 </td><td></td></tr><tr><td> COT-1 DNA 1毫克/毫升</td><td> Invitrogen公司</td><td> ＃18440 </td><td></td></tr><tr><td> hybloc DNA的1毫克/毫升</td><td>应用遗传</td><td> ＃MHB </td><td></td></tr><tr><td>鲑鱼精</td><td>西格玛</td><td> ＃D1626 </td><td>粉末的再悬浮在H 2 O在10毫克/毫升</td></tr><tr><td>乙酸钠（醋酸钠，pH值5.2，缓冲溶液） </td><td>西格玛</td><td> ＃S7899 </td><td></td></tr><tr><td> FICOLL 400（分子生物学级） </td><td>费舍尔</td><td>排名第525 </td><td></td></tr><tr><td>聚乙烯吡咯烷酮（分子生物学级） </td><td>费舍尔</td><td> ＃BP431 </td><td></td></tr><tr><td>硫酸葡聚糖粉末</td><td>西格玛</td><td> ＃D8906 </td><td></td></tr><tr><td>亚急性硬化性全脑炎（盐磷酸钠EDTA）20倍的解决方案</td&gt; <td>费舍尔</td><td> ＃BP1328 </td><td></td></tr><tr><td>甲酰胺</td><td>费舍尔</td><td> ＃BP227 </td><td></td></tr><tr><td>盖玻片</td><td>费舍尔</td><td> ＃12-548-B </td><td></td></tr><tr><td>幻灯片</td><td>费舍尔</td><td> ＃12-550 </td><td></td></tr><tr><td> 6 - 孔板</td><td>费舍尔</td><td> ＃0720080 </td><td></td></tr><tr><td> PBS，10 </td><td>费舍尔</td><td> ＃MT-46-013-CM </td><td></td></tr><tr><td>聚-L-赖氨酸水溶液</td><td>西格玛</td><td> ＃P8920 </td><td></td></tr><tr><td>多聚甲醛，小球状，95％; </td><td>西格玛</td><td> ＃441244 </td><td></td></tr><tr><td>的Triton-X-100，分子生物学级</td><td>西格玛</td><td> ＃T8787 </td><td></td></tr><tr><td> BSA（牛血清白蛋白）馏分V </td><td>费舍尔</td><td> ＃BP 1600 </td><td></td></tr><tr><td>正常山羊血清</td><td>矢量实验室</td><td> ＃S-1000 </td><td></td></tr><tr><td>吐温20，分子生物学级</td><td>西格玛</td><td> ＃P9416 </td><td></td></tr><tr><td>的SSC（盐柠檬酸钠）20倍的解决方案</td><td>费舍尔</td><td> ＃BP1325 </td><td></td></tr><tr><td>延长Gold抗淬灭试剂</td><td> Invitrogen公司</td><td> ＃P36930 </td><td></td></tr><tr><td> DAPI（4&#39;，6 - 二脒基-2 - 苯基吲哚） </td><td>西格玛</td><td> ＃D9542 </td><td></td></tr><tr><td>最好的测试涂层橡胶水泥</td><td>艺术或办公用品店</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> 表1。特定的试剂和小型设备。 </td></tr></tbody></table>

combined immunofluorescence and dna fish on 3d-preserved interphase nuclei to study changes in 3d nuclear organization

荧光原位杂交DNA探针完好的三维保存的核三维激光共聚焦显微镜（3D DNA FISH）表示，最直接的方法来可视化基因位点的位置，染色体的次区域或整个地区的单个细胞。这种类型的分析提供洞察全球架构的细胞核，以及特定的基因组位点的行为和地区内的核空间。免疫荧光法，另一方面，允许核蛋白质（修饰组蛋白，组蛋白变体和改性剂，转录机械因素，核分舱室，等）的检测。免疫荧光和3D的DNA结合FISH的主要挑战是，一方面要保留的表位，以及由抗体检测的细胞核的3D架构上，另一方面，允许渗透的DNA探针来检测基因位点或染色体地区1-5。在这里，我们提供了一个协议，它结合了可视化的三维保存的核染色质修饰与基因位点在。

DNA和免疫-FISH用于的Skok实验室，研究的过程中，V（D）J重组抗原受体位点在B和T淋巴细胞的发展与核组织的变化。上面详述的技术使我们ⅱ）到i）之间的测量距离的两个端部的轨迹（收缩）之间的测量距离的等位基因或基因座（配对），iii）分析内发生的位点的DNA损伤，ⅳ）评估的等位基因的位置位点相对于核分室（在我们的研究的镇压着丝粒异染色质），及v）评估协会的细胞核蛋白的基因和?...

Watch this Scientific Journal Video about Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization at JoVE.com

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

在这里，我们描述了一个协议，用于同时检测组蛋白修饰免疫荧光法和DNA序列的DNA荧光原位杂交，然后通过的3D显微镜和分析（3D的免疫-DNA FISH）。

结合免疫荧光和三维保存的间期核荧光原位杂交DNA研究在3D核组织的变化

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

combined-immunofluorescence-dna-fish-on-3d-preserved-interphase

Research

JoVE Journal

Biology

18.2K 次观看.  New York University School of Medicine. 在这里，我们描述了一个协议，用于同时检测组蛋白修饰免疫荧光法和DNA序列的DNA荧光原位杂交，然后通过的3D显微镜和分析（3D的免疫-DNA FISH）。

视频: Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

DNA-based Fish Species Identification Protocol

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. This versatile method employs PCR amplification of genomic DNA extracted from fish samples, followed by restriction fragment length polymorphism (RFLP) analysis to generate fragment patterns that can be resolved on the Agilent 2100 Bioanalyzer and matched to the correct species using RFLP pattern matching software.
The fish identification method uses a simple, reliable, spin column- based protocol to isolate DNA from fish samples. The samples are treated with proteinase K to release the nucleic acids into solution. DNA is then isolated by suspending the sample in binding buffer and loading onto a micro- spin cup containing a silica- based fiber matrix. The nucleic acids in the sample bind to the fiber matrix. The immobilized nucleic acids are washed to remove contaminants, and total DNA is recovered in a final volume of 100 &mu;l. The isolated DNA is ready for PCR amplification with the provided primers that bind to sequences found in all fish genomes. The PCR products are then digested with three different restriction enzymes and resolved on the Agilent 2100 Bioanalyzer. The fragment lengths produced in the digestion reactions can be used to determine the species of fish from which the DNA sample was prepared, using the RFLP pattern matching software containing a database of experimentally- derived RFLP patterns from commercially relevant fish species.

This publication describes how to use the Agilent Fish Species Identification System to identify the species of a fish by extracting DNA and performing PCR and RFLP analysis.

以DNA为基础的鱼类物种鉴定协议

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. ...

Robust 3D DNA FISH Using Directly Labeled Probes

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes.

强大的3D直接标记的探针DNA鱼

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to the histological and morphological development of vertebrate embryos. In particular, the use of chimeric embryos has established the importance of neural crest in directing the species-specific morphology of the craniofacial complex. The method described herein utilizes two avian species, duck and quail, with remarkably different craniofacial morphology. This method greatly facilitates the investigation of molecular and cellular regulation of species-specific pattern in the craniofacial complex.&#160;Experiments in quail and duck chimeric embryos have already revealed neural crest-mediated tissue interactions and cell-autonomous behaviors that regulate species-specific pattern in the craniofacial skeleton, musculature, and integument. The great diversity of neural crest derivatives suggests significant potential for future applications of the quail-duck chimeric system to understanding vertebrate development, disease, and evolution.

This article describes a method to generate chimeric embryos that are&#160;designed to test the species-specific contributions of neural crest and/or other tissues to craniofacial development.

评估具体物种的贡献颅面开发利用鹌鹑，鸭嵌合体

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to ...

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

结合DNA-RNA荧光<em&gt;原位</em&gt;杂交（FISH）研究X染色体失活的分化雌​​性小鼠胚胎干细胞

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high radiation doses. Addressing questions related to environmental, occupational and public health safety in a proper and scientifically justified manner heavily relies on the ability to accurately measure the biological effects of low dose pollutants, such as ionizing radiation and chemical substances. DNA damage and repair are the most important early indicators of health risks due to their potential long term consequences, such as cancer. Here we describe a protocol to study the effect of chronic in vivo exposure to low doses of &#947;- and &#946;-radiation on DNA damage and repair in mouse spleen cells. Using a commonly accepted marker of DNA double-strand breaks, phosphorylated histone H2AX called &#947;H2AX, we demonstrate how it can be used to evaluate not only the levels of DNA damage, but also changes in the DNA repair capacity potentially produced by low dose in vivo exposures. Flow cytometry allows fast, accurate and reliable measurement of immunofluorescently labeled &#947;H2AX in a large number of samples. DNA double-strand break repair can be evaluated by exposing extracted splenocytes to a challenging dose of 2 Gy to produce a sufficient number of DNA breaks to trigger repair and by measuring the induced (1 hr post-irradiation) and residual DNA damage (24 hrs post-irradiation). Residual DNA damage would be indicative of incomplete repair and the risk of long-term genomic instability and cancer. Combined with other assays and end-points that can easily be measured in such in vivo studies (e.g., chromosomal aberrations, micronuclei frequencies in bone marrow reticulocytes, gene expression, etc.), this approach allows an accurate and contextual evaluation of the biological effects of low level stressors.

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

A protocol to evaluate changes in DNA damage levels and DNA repair capacity that may be induced by chronic in vivo low dose irradiation in mouse spleen lymphocytes, by measuring phosphorylated histone H2AX, a marker of DNA double-strand breaks, using flow cytometry is presented.

测量DNA损伤与修复小鼠脾细胞慢性后<em&gt;在体内</em&gt;暴露于β和γ辐射剂量很低

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

具体的基因组区域及相关分子鉴定enChIP隔离

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

方法研究线虫线虫中固有的蛋白质聚集与年龄的变化

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Sequential Immunofluorescence and Immunohistochemistry on Cryosectioned Zebrafish Embryos

Investigation of intercellular interactions often requires discrete labeling of specific cell populations and precise protein localization. The zebrafish embryo is an excellent tool for examining such interactions with an in vivo model. Whole-mount immunohistochemical and immunofluorescence assays are frequently applied in zebrafish embryos to assess protein expression. However, it can be difficult to achieve accurate mapping of co-localized proteins in three-dimensional space. In addition, some studies may require the use of two antibodies that are not compatible with the same technique (e.g., antibody 1 is only suitable for immunohistochemistry and antibody 2 is only suitable for immunofluorescence). The purpose of the method described herein is to perform sequential immunofluorescence and/or immunohistochemistry on individual cryosections derived from early-stage zebrafish embryos. Here we describe the use of sequential rounds of immunofluorescence, imaging, immunohistochemistry, imaging for a single cryosection in order to achieve precise identification of protein expression at the single-cell level. This methodology is suitable for any study in early-stage zebrafish embryos that requires accurate identification of multiple protein targets in individual cells.

This protocol demonstrates sequential immunofluorescence and immunohistochemistry on cryosections from early-stage zebrafish embryos enabling precise colocalization analyses in specific cell populations.

冷冻斑马鱼胚胎的连续免疫荧光与免疫组化学

Investigation of intercellular interactions often requires discrete labeling of specific cell populations and precise protein localization. The zebrafish ...

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance.
There are two major ways of visualizing&#160;proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or &#8220;foci&#8221;, that can be visualized by indirect immunofluorescence using specific antibodies.
Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.