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

Nous tenons à remercier les membres du laboratoire Skok, surtout Susannah Hewitt, de discussions et de commentaires. Ce travail est soutenu par l&#39;Institut national de la santé subventions R01GM086852, RC1CA145746 (JAS). JAS est une leucémie et érudit Lymphoma Society. JC est un Fellow Irvington Institut de la recherche sur le cancer Institut. MM est soutenu par une éducation National Science Foundation Grant universitaire intégré et des stages de recherche (NSF IGERT 0333389).

1. Étiquetage sonde d&#39;ADN avec des fluorophores: Nick Translation (~ 6 h) <ol><li> Propre ADN de BAC (préparé par maxi-prep) ou des plasmides ou des produits de PCR, tous remis en suspension dans H 2 O, peut être utilisé pour le marquage. Notez que pour un signal FISH robuste, sondes doivent couvrir au moins 10 kb. </li><li> Incuber ADN dans RNase A pendant 30 min à 37 ° C (Tous les réactifs sont énumérées dans le tableau 1). </li><li> Incuber la réaction tr...

<ol>
	<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>

Les techniques décrites ci-dessus ont été utilisées dans notre laboratoire pour analyser la réglementation de V (D) J de l&#39;immunoglobuline et TCRA / j loci dans le développement des lymphocytes 30,31. Nous sommes convaincus que cette technique peut être adaptée pour la détection de diverses protéines nucléaires, des compartiments nucléaires et les lieux, dans différents types cellulaires. Modifications du protocole peut être nécessaire, et dans ce cas les étapes majeures ...

Épigénétique mise en place de mécanismes de déclenchement et l&#39;héritage de développement et d&#39;un type de cellule spécifique profils transcriptionnels. À un certain niveau, cela implique la modulation de empaquetage de la chromatine qui définit des régions génomiques actifs ou silencieux. À plus grande échelle, l&#39;organisation mondiale de la 3D et de l&#39;architecture du génome nucléaire jouent également un rôle dans le contrôle des motifs de la transcription. Ainsi, la dissection de ces caractéristiques épigénomiques est essentielle pour une compréhension complète de la façon dont les gènes sont régulés 6-11. Combiné FISH ADN immunofluorescence et 3D offrent une occasion unique de compléter les analyses moléculaires et biochimiques en évaluant les interactions spécifiques / associations de séquences d&#39;ADN et / ou de protéines dans le noyau. En outre, alors que l&#39;ensemble du génome techniques à haut débit tels que immunoprécipitation de la chromatine (ChIP-seq) ou la conformation de capture chromosome couplé avec le séquençage profond (4C-seq, 5C, Salut-C) fournir dat mondialeun sur 12 populations de cellules, les techniques d&#39;immunofluorescence POISSON / ADN permettre des analyses au niveau de la cellule unique. Nous décrivons ici un protocole pour la détection simultanée des modifications des histones par des séquences d&#39;ADN immunofluorescence et par FISH ADN suivies par microscopie 3D et des analyses (3D immuno-FISH). L&#39;avantage de ce protocole est la visualisation combinée de l&#39;ADN et la préservation de la structure des protéines. Notre expérience dans ce domaine nous a permis d&#39;améliorer et de simplifier les protocoles existants. Bien que nous ayons utilisé ce protocole pour détecter l&#39;ADN des cassures double brin dans les lymphocytes subissent une recombinaison, cette méthode peut être appliquée à d&#39;autres protéines et d&#39;autres types de cellules.

<table border="1"><tbody><tr><td> Nom de réactif / Matériel </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> H 2 O </td><td> Pêcheur </td><td> # BP2470 </td><td></td></tr><tr><td> RNase A </td><td> Sigma </td><td> # R4642 </td><td></td></tr><tr><td> dNTP </td><td> Sigma </td><td> # DNTP100 </td><td></td></tr><tr><td> Alexa dUTP </td><td> Invitrogen </td><td> # C11397 à C-11401 </td><td></td></tr><tr><td> Cy3 ou Cy5 dUTP </td><td> Pêcheur </td><td> # 45-001-xxx </td><td></td></tr><tr><td> DNase I </td><td> Roche </td><td> # 04536282001 </td><td></td></tr><tr><td> ADN Pol I </td><td> Biolabs </td><td> # M0209 </td><td></td></tr><tr><td> 0.025 filtres pm </td><td> Millipore </td><td> # VSWP02500 </td><td></td></tr><tr><td> ADN Cot-1 1 mg / ml </td><td> Invitrogen </td><td> # 18440 </td><td></td></tr><tr><td> Hybloc ADN 1 mg / ml </td><td> Applied Genetics </td><td> # MHB </td><td></td></tr><tr><td> De sperme de saumon </td><td> Sigma </td><td> # D1626 </td><td> poudre pour être remises en suspension à 10 mg / ml dans du H 2 O </td></tr><tr><td> NaAc (acétate de sodium, pH 5,2, solution tampon) </td><td> Sigma </td><td> # S7899 </td><td></td></tr><tr><td> Ficoll 400 (Mol Biol grade) </td><td> Pêcheur </td><td> N ° 525 </td><td></td></tr><tr><td> Polyvinylpyrrolidone (Mol Biol grade) </td><td> Pêcheur </td><td> # BP431 </td><td></td></tr><tr><td> Poudre de sulfate de dextrane </td><td> Sigma </td><td> # D8906 </td><td></td></tr><tr><td> PESS (Saline-phosphate de sodium-EDTA) solution 20x </td&gt; <td> Pêcheur </td><td> # BP1328 </td><td></td></tr><tr><td> Formamide </td><td> Pêcheur </td><td> # BP227 </td><td></td></tr><tr><td> Lamelles de </td><td> Pêcheur </td><td> N ° 12-548-B </td><td></td></tr><tr><td> Diapositives </td><td> Pêcheur </td><td> # 12-550 </td><td></td></tr><tr><td> Plaques de 6 puits </td><td> Pêcheur </td><td> # 0720080 </td><td></td></tr><tr><td> PBS, 10x </td><td> Pêcheur </td><td> # MT-46-013-CM </td><td></td></tr><tr><td> La poly-L-lysine solution </td><td> Sigma </td><td> # P8920 </td><td></td></tr><tr><td> Paraformaldéhyde, granulés, 95% </td><td> Sigma </td><td> # 441244 </td><td></td></tr><tr><td> Triton-X-100, Mol Biol qualité </td><td> Sigma </td><td> # T8787 </td><td></td></tr><tr><td> Fraction BSA (Bovine Serum Albumin) V </td><td> Pêcheur </td><td> # BP 1600 </td><td></td></tr><tr><td> Sérum normal de chèvre </td><td> Vector Labs </td><td> # S-1000 </td><td></td></tr><tr><td> Tween-20, Mol Biol qualité </td><td> Sigma </td><td> # P9416 </td><td></td></tr><tr><td> SSC (citrate de sodium salin) 20x solution </td><td> Pêcheur </td><td> # BP1325 </td><td></td></tr><tr><td> ProLong réactif antifade Or </td><td> Invitrogen </td><td> # P36930 </td><td></td></tr><tr><td> DAPI (4 &#39;,6-diamidino-2-phénylindole) </td><td> Sigma </td><td> # D9542 </td><td></td></tr><tr><td> Meilleur essai un ciment enveloppe en caoutchouc </td><td> Magasins de fournitures d&#39;art ou de bureau </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> Tableau 1. Réactifs spécifiques et du petit matériel. </td></tr></tbody></table>

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

Hybridation fluorescente in situ utilisant des sondes ADN sur le 3-dimensions noyaux conservés suivie par microscopie confocale 3D (FISH ADN 3D) représente le moyen le plus direct pour visualiser l&#39;emplacement des loci de gènes, chromosomes sous-régions ou des territoires entiers dans des cellules individuelles. Ce type d&#39;analyse permet de mieux comprendre l&#39;architecture globale du noyau ainsi que le comportement des loci génomiques spécifiques et des régions dans l&#39;espace nucléaire. Immunofluorescence, d&#39;autre part, permet la détection des protéines nucléaires (histones modifiées, variants d&#39;histones et des modificateurs, des machines et des facteurs de transcription nucléaires, sous-compartiments, etc.) Le problème majeur en combinant FISH ADN immunofluorescence et 3D est, d&#39;une part, de préserver l&#39;épitope détecté par l&#39;anticorps, ainsi que l&#39;architecture 3D du noyau, et d&#39;autre part, pour permettre la pénétration de la sonde d&#39;ADN pour détecter loci chromosomiques ou territoires 1-5. Ici, nous fournissons un protocole qui combine la visualisation des modifications de la chromatine avec loci génomiques conservées dans les noyaux 3D.

ADN et immuno-FISH sont utilisés dans le laboratoire Skok pour étudier les changements dans l&#39;organisation nucléaire associés au processus de V (D) J du locus récepteurs des antigènes au cours du développement des lymphocytes B et T. Les techniques décrites ci-dessus nous permettent de mesurer des distances i) entre les deux extrémités d&#39;un locus (contraction) ii) mesurer les distances entre les allèles ou loci (appariement), iii) analyser l&#39;endommagement de l&#39;ADN survenant dans les lieux, iv)...

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

Nous décrivons ici un protocole pour la détection simultanée des modifications des histones par des séquences d&#39;ADN immunofluorescence et par FISH ADN suivies par microscopie 3D (3D et des analyses immuno-FISH ADN).

Immunofluorescence combinés et FISH ADN sur 3D préservé noyaux en interphase pour étudier les changements dans l&#39;organisation nucléaire 3D

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

Test sensibilité visuelle à la vitesse et la direction du mouvement chez les lézards

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

Recherche

Biologie

DNA-based Fish Species Identification Protocol

Basées sur l&#39;ADN des espèces de poissons protocole d&#39;identification

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

Robuste FISH 3D DNA Utilisation des sondes directement marquées

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

Évaluation des contributions spécifiques aux espèces Vers développement cranio-facial Utilisation chimères caille-canard

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

ADN-ARN combiné Fluorescent<em&gt; In situ</em&gt; Hybridation (FISH) à étudier inactivation du chromosome X dans différencié Femme Souris cellules souches embryonnaires

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

Mesure de dommages et réparation de l&#39;ADN dans les splénocytes de souris Après chronique<em&gt; In Vivo</em&gt; L&#39;exposition à de très faibles doses de bêta et gamma Rayonnement

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

Approches expérimentales pour l&#39;étude mitochondrial Localisation et fonction d&#39;une cellule du cycle nucléaire Kinase, Cdk1

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

Isolement des régions génomiques spécifiques et l&#39;identification des molécules associées par enChIP

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

Méthodes d’étude des changements dans l’agrégation des protéines inhérente avec l’âge chez 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 ...

Sequential Immunofluorescence and Immunohistochemistry on Cryosectioned Zebrafish Embryos

Immunofluorescence séquentielle et immunohistochimie sur les embryons de poissons zèbres cryosectionnés

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