Name: combined immunofluorescence and dna fish on 3d-preserved interphase nuclei to study changes in 3d nuclear organization
Uploaded: 2013-02-03T14:00:00
Description: 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

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?...

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	<li>Chaumeil, J., Okamoto, I., 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=X-chromosome+inactivation+in+mouse+embryonic+stem+cells:+analysis+of+histone+modifications+and+transcriptional+activity+using+immunofluorescence+and+FISH.">X-chromosome inactivation in mouse embryonic stem cells: analysis of histone modifications and transcriptional activity using immunofluorescence and FISH.</a> Methods in enzymology. , 376-405 (2004).</li><li>Cremer, M., 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=Multicolor+3D+fluorescence+in+situ+hybridization+for+imaging+interphase+chromosomes.">Multicolor 3D fluorescence in situ hybridization for imaging interphase chromosomes.</a> Methods Mol. Biol. 463, 205-239 (2008).</li><li>Chaumeil, J., Augui, S., Chow, J. C., 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=Combined+immunofluorescence,+RNA+fluorescent+in+situ+hybridization,+and+DNA+fluorescent+in+situ+hybridization+to+study+chromatin+changes,+transcriptional+activity,+nuclear+organization,+and+X-chromosome+inactivation.">Combined immunofluorescence, RNA fluorescent in situ hybridization, and DNA fluorescent in situ hybridization to study chromatin changes, transcriptional activity, nuclear organization, and X-chromosome inactivation.</a> Methods Mol. Biol. 463, 297-308 (2008).</li><li>Solovei, I., Cremer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D-FISH+on+cultured+cells+combined+with+immunostaining.">3D-FISH on cultured cells combined with immunostaining.</a> Methods Mol. Biol. 659, 117-126 (2010).</li><li>Markaki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+3D-FISH+and+super-resolution+structured+illumination+microscopy+for+studies+of+3D+nuclear+architecture:+3D+structured+illumination+microscopy+of+defined+chromosomal+structures+visualized+by+3D+(immuno)-FISH+opens+new+perspectives+for+studies+of+nuclear+architecture.">The potential of 3D-FISH and super-resolution structured illumination microscopy for studies of 3D nuclear architecture: 3D structured illumination microscopy of defined chromosomal structures visualized by 3D (immuno)-FISH opens new perspectives for studies of nuclear architecture.</a> BioEssays : news and reviews in molecular, cellular and developmental biology. 34, 412-426 (2012).</li><li>Heard, E., Bickmore, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ins+and+outs+of+gene+regulation+and+chromosome+territory+organisation.">The ins and outs of gene regulation and chromosome territory organisation.</a> Current opinion in cell biology. 19, 311-316 (2007).</li><li>Misteli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+the+sequence:+cellular+organization+of+genome+function.">Beyond the sequence: cellular organization of genome function.</a> Cell. 128, 787-800 (1016).</li><li>Fraser, P., Bickmore, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+organization+of+the+genome+and+the+potential+for+gene+regulation.">Nuclear organization of the genome and the potential for gene regulation.</a> Nature. 447, 413-417 (2007).</li><li>Cremer, T., 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=Chromosome+territories--a+functional+nuclear+landscape.">Chromosome territories--a functional nuclear landscape.</a> Current opinion in cell biology. 18, 307-316 (2006).</li><li>Mao, Y. S., Zhang, B., Spector, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis+and+function+of+nuclear+bodies.">Biogenesis and function of nuclear bodies.</a> Trends in genetics : TIG. 27, 295-306 (2011).</li><li>Dostie, J., 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=Chromosome+organization+in+the+nucleus+-+charting+new+territory+across+the+Hi-Cs.">Chromosome organization in the nucleus - charting new territory across the Hi-Cs.</a> Current opinion in genetics &amp; development. 22, 125-131 (2012).</li><li>van Steensel, B., Dekker, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomics+tools+for+unraveling+chromosome+architecture.">Genomics tools for unraveling chromosome architecture.</a> Nature. 28, 1089-1095 (2010).</li><li>Massey, F. 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+Kolmogorov-Smirnov+Test+for+Goodness+of+Fit.">The Kolmogorov-Smirnov Test for Goodness of Fit.</a> Journal of the American Statistical Association. 253, 1951 (1951).</li><li>Collins, 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=RUNX+transcription+factor-mediated+association+of+Cd4+and+Cd8+enables+coordinate+gene+regulation.">RUNX transcription factor-mediated association of Cd4 and Cd8 enables coordinate gene regulation.</a> Immunity. 34, 303-314 (2011).</li><li>Fisher, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+interpretation+of+&#967;2+from+contingency+tables,+and+the+calculation+of+P.">On the interpretation of &#967;2 from contingency tables, and the calculation of P.</a> Journal of the Royal Statistical Society. 85, 87-94 (1922).</li><li>Benjamini, Y. H., Yosef, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+the+false+discovery+rate:+a+practical+and+powerful+approach+to+multiple+testing.">Controlling the false discovery rate: a practical and powerful approach to multiple testing.</a> Journal of the Royal Statistical Society, Series B (Methodological). 57, 125-133 (1995).</li><li>Fitzsimmons, S. P., Bernstein, R. M., Max, E. E., Skok, J. A., Shapiro, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+changes+in+accessibility,+nuclear+positioning,+recombination,+and+transcription+at+the+Igkappa+locus.">Dynamic changes in accessibility, nuclear positioning, recombination, and transcription at the Igkappa locus.</a> J. Immunol. 179, 5264-5273 (2007).</li><li>Fuxa, M., 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=Pax5+induces+V-to-DJ+rearrangements+and+locus+contraction+of+the+immunoglobulin+heavy-chain+gene.">Pax5 induces V-to-DJ rearrangements and locus contraction of the immunoglobulin heavy-chain gene.</a> Genes Dev. 18, 411-422 (2004).</li><li>Goldmit, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+ontogeny+of+the+Igk+locus+during+B+cell+development.">Epigenetic ontogeny of the Igk locus during B cell development.</a> Nature. 6, 198-203 (2005).</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=Association+between+the+Igk+and+Igh+immunoglobulin+loci+mediated+by+the+3'+Igk+enhancer+induces+'decontraction'+of+the+Igh+locus+in+pre-B+cells.">Association between the Igk and Igh immunoglobulin loci mediated by the 3' Igk enhancer induces 'decontraction' of the Igh locus in pre-B cells.</a> Nature. 9, 396-404 (2008).</li><li>Johnson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-7+Functionally+Segregates+the+Pro-B+Cell+Stage+by+Regulating+Transcription+of+Recombination+Mediators+across+Cell+Cycle.">IL-7 Functionally Segregates the Pro-B Cell Stage by Regulating Transcription of Recombination Mediators across Cell Cycle.</a> Journal of Immunology. , (2012).</li><li>Karnowski, 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=Silencing+and+nuclear+repositioning+of+the+lambda5+gene+locus+at+the+pre-b+cell+stage+requires+Aiolos+and+OBF-1.">Silencing and nuclear repositioning of the lambda5 gene locus at the pre-b cell stage requires Aiolos and OBF-1.</a> PLoS ONE. 3, e3568 (2008).</li><li>Kosak, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subnuclear+compartmentalization+of+immunoglobulin+loci+during+lymphocyte+development.">Subnuclear compartmentalization of immunoglobulin loci during lymphocyte development.</a> Science. 296, 158-162 (2002).</li><li>Liu, H., 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=Yin+Yang+1+is+a+critical+regulator+of+B-cell+development.">Yin Yang 1 is a critical regulator of B-cell development.</a> Genes Dev. 21, 1179-1189 (2007).</li><li>Parker, 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=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>

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 ...

Research

JoVE Journal

Biology

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

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

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

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

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

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

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

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 ...

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

实验方法来研究线粒体定位和核细胞周期激酶CDK1功能

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The ...

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

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

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

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

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 ...