Gostaríamos de agradecer aos membros do laboratório Skok, especialmente Susannah Hewitt, para discussões e comentários. Este trabalho é apoiado pelo Instituto Nacional de bolsas de Saúde R01GM086852, RC1CA145746 (JAS). JAS é um Leukemia &amp; Lymphoma Society estudioso. JC é um Irvington Fellow Instituto do Cancer Research Institute. MM é apoiado por um National Science Foundation Grant Integrativa Pós-Graduação Pesquisa e Estágio (NSF IGERT 0.333.389).

1. DNA Labeling Sonda com fluoróforos: Nick Tradução (~ 6 horas) <ol><li> Limpeza BAC DNA (preparado por maxi-prep) ou plasmídeos ou produtos de PCR, todos ressuspenso em H 2 O, podem ser utilizados para a rotulagem. Note-se que para um sinal de FISH robusto, as sondas devem abranger pelo menos 10 kb. </li><li> Incubar DNA em RNase A durante 30 min a 37 ° C (todos os reagentes estão listados na Tabela 1). </li><li> Incubar a reacção de tradução de nick durante 2 horas a 16 ° C (ver Tabelas 2 e 3). Os métodos alternativos de marcação directa pode ser utilizado (exemplo: Tag FISH, Invitrogen). </li><li> Inactivar a reacção durante uma hora à temperatura de -80 ° C ou durante a noite a -20 ° C. </li><li> Determinar o tamanho da sonda sobre um gel de agarose a 2%. O esfregaço deve estar entre 100 e 1000 pb, com a maioria a cerca de 300-500 pb (Note-se que a mancha de Cy5 sondas não é visível). Se o DNA não é digerida suficiente, mais DNA Pol I eDNase I pode ser adicionado à mistura reaccional e incubou-se durante mais duas horas a 16 ° C. </li><li> Purifica-se as sondas de 0,025 milímetros filtros em duas provetas de litros cheio com H 2 0 por duas horas no escuro. </li><li> Adicionar factores de bloqueio para as sondas como se segue: 10 ug de ADN Cot-1, 10 ug de DNA Hybloc e 100 ug de esperma de salmão de 10 ^ g de sondas de DNA. Loja de sondas de ADN a -20 ° C. </li></ol> 2. Probe Precipitation / desnaturação / pré-hibridação (3-4 horas) <ol><li> Entre 0,3 ug e 1 ug de DNA de sonda é usada por lamela. Sondas de mistura com 0,1 volumes de NaAc 3M (pH 5,2) e 2,5 volumes de etanol a 100%, a incubação durante uma hora a -80 ° C ou durante a noite a -20 ° C e centrifugação a 13.000 rpm para baixo a 4 ° C durante 30 min. Note-se que a quantidade de sonda de DNA pode ser ajustada em função da qualidade do DNA e da região nuclear de hibridação. Os métodos alternativos de marcação directa podem permitir a utilização de quantidades reduzidas desonda. Para a detecção de sinais de DNA várias peixe em um slide, as diferentes sondas pode ser precipitada juntos (exceto sondas que não deve ser pré-recozido, como seqüências de repetição). Sondas para várias experiências realizadas ao mesmo tempo, podem também ser precipitados juntos. Pellets deve ser levemente coloridos de acordo com os fluoróforos usados ​​(se pequenas quantidades de sondas são precipitados, o sedimento pode não ser visível). </li><li> Pelotas secas e ressuspender-los em 15 ul de tampão de hibridação (ver Tabela 4) por cada lamela com agitação (usando um Thermomixer de Eppendorf) no escuro a 37-45 ° C (durante cerca de 20 min). </li><li> Sondas desnaturar durante 5 min a 75-95 ° C. </li><li> Pré-recozer sondas, a 37 ° C durante 30-60 min. Comprimento de pré-hibridação pode ser ajustada em função da complexidade e da qualidade das sondas. </li><li> Aplicar sondas para as lamelas para cruzamentos durante a noite (ver Seção 3). </li></ol> 3. 3PEIXES-dimensional DNA - Primeiro Dia (~ 3 horas) <ol><li> As células não aderentes são concentrados num pequeno volume de 1x PBS (~ 2x10 5 células em 5-10 ul de PBS 1x por lamela) e deixado cair no centro de uma lamela de poli-L-lisina revestido. As células aderentes deverá ser cultivadas em lamelas durante pelo menos 24 h (~ 70% de confluência) (lamelas podem ser revestidos com gelatina a 0,1%). Quadrados 18x18 para lamelas 24-24 mm são colocados em placas de 6 poços, e ao longo do protocolo, dois ml de cada solução é usada por poço (alternativamente redondo 1,5 mm, podem ser lamelas colocadas em placas de 12/24-well). </li><li> Fixar as células em paraformaldeído a 2% / PBS 1x, pH 7-7,4, durante 10 min à TA (stocks PFA a 4% pode ser dividido em alíquotas, armazenado a -20 ° C). Paraformaldeído podem ser comprados em solução (16%), ou as soluções de reserva de 4% pode ser preparada a partir de pó / grânulos. Paraformaldeído em pó é dissolvido em 1x PBS a&gt; 60 ° C (pH pode ser aumentado para 8-9 para facilitar a dissolução), arrefecido para baixo sobre o gelo, ajustared a pH 7-7,4, filtrada, aliquotadas e armazenadas em falcões durante semanas à temperatura de -20 ° C. </li><li> Após três lavagens em PBS 1x, permeabilizar as células em gelado fria de 0,4% de Triton-X-100/1 x PBS durante 5 min em gelo. Comprimento de permeabilização pode ser ajustado dependendo do tipo de célula-NB:. PEIXES Se o DNA é combinado com imunofluorescência, a imunomarcação devem ser realizados nesta fase (ver secção 4). </li><li> Após três lavagens em PBS 1x, as células a incubar com 0,1 ug / ul de ARNase A em 1x PBS, durante uma hora a 37 ° C. As lamelas são colocadas lado a célula para baixo sobre uma gota de 100 ul de solução em parafilme ou uma lâmina em câmara húmida. As lamelas são em seguida cuidadosamente removida com uma pinça. Se alguma resistência é encontrada, lamelas devem ser inundado com 1x PBS, a fim de evitar danificar as células. </li><li> Após três lavagens em PBS 1x, permeabilizar as células em gelado fria de 0,7% de Triton-X-100 / HCl 0,1 M durante 10 minutos em gelo. </li><li> Após três lavagens in 1x PBS, as células desnaturam em HCl 1,9 M durante 30 min à temperatura ambiente. Alternativamente, as células podem ser desnaturadas em formamida 50% / 2x SSC a 75-80 ° C durante pelo menos 30 min. Note-se que a hibridação comprimento (e da temperatura), pode ser optimizada, dependendo do tipo de célula. </li><li> Após três lavagens em gelo frio 1x PBS, hibridar com sondas de células durante a noite a 37 ° C numa câmara escura e húmida. Lamelas são colocados lado a célula para baixo em uma queda de 15 ul de sonda em um slide, e selado com cimento de borracha. </li></ol> 4. 3-dimensional Imuno-FISH - Primeiro dia (~ 6-7 horas) <ol><li> Por combinados FISH imunofluorescência / DNA, imuno-coloração deve ser realizada após a permeabilização em 0,4% de Triton-X-100 (Passo 3.3). </li><li> Incubar as células em solução de bloqueio durante 30 minutos a RT (2,5% de albumina sérica bovina (BSA), soro de cabra normal a 10%, 0,1% de Tween-20). A solução de bloqueio é filtrado, aliquotadas em 1,5 ml eppendorfs e armazenado para meses a -20 ° C. </li><li> Incubar as células com o anticorpo primário produzido contra a proteína ou a modificação das histonas de interesse diluído em solução de bloqueio, por uma hora, à temperatura ambiente numa câmara húmida. Aqui, mostramos o exemplo de um anticorpo contra a serina-139 fosforilada da H2AX (γ-H2AX, Millipore; Consulte a Tabela 5 para detalhes). As lamelas são colocadas lado a célula para baixo sobre uma gota de 100 ul de solução em parafilme ou uma lâmina em câmara húmida. As lamelas são em seguida cuidadosamente removida com uma pinça (inundado com 1x PBS, se necessário). Note-se que a diluição e tempo de incubação pode ser ajustada em função da qualidade do anticorpo e tipos de células. </li><li> Lave as células em 0,2% BSA / 0,1% de Tween-20/1 x PBS, três vezes 5 min à temperatura ambiente com agitação. </li><li> Incubar as células com o anticorpo conjugado com fluoróforo adequado secundário diluído em solução de bloqueio durante uma hora, à temperatura ambiente numa câmara escura e húmida. Aqui vamos mostrar um exemplo de detecção com umasecundário anticorpo de cabra anti-rato (Alexa Fluor 488 ou 555, Invitrogen; Consulte a Tabela 5 para detalhes). Hapteno-anticorpos secundários conjugados (biotina, digoxigenina, etc) podem também ser usados. Neste caso, um passo adicional para a detecção apropriada (estreptavidina, anti-dig, etc) pode ser realizada antes ou depois durante a noite a hibridação de DNA-FISH. </li><li> Enxaguar as células em 0,1% de Tween-20/1 x PBS, três vezes 5 min à temperatura ambiente com agitação no escuro. </li><li> Pós-fix células em paraformaldeído a 2% / PBS 1x durante 10 min à RT, no escuro. </li><li> Continuar FISH DNA como acima de incubação em RNase A (Passo 3.4). </li></ol> 5. 3-dimensional DNA FISH / Imuno-FISH - Segundo dia (~ 2 horas) <ol><li> Remova cuidadosamente cimento de borracha, lamínulas de inundação com 2x SSC, remova cuidadosamente com uma pinça e colocá-los em poços contendo solução de lavagem. </li><li> Lave as células em 2x SSC durante 30 min a 37 ° C no escuro, com agitação.</li><li> Lave as células em 2x SSC durante 30 min à RT, no escuro, com agitação. </li><li> Lave as células em 1x SSC durante 30 min à RT, no escuro, com agitação Note-se que em caso de desnaturação com formamida, lavagens deve ser substituído pelo seguinte:. Lavagens três lavagens em 50% de formamida / SSC 2x e 3 em 2x SSC, 5 min cada, a 37 ° C. </li><li> Montar as células em meio de prolongar a ouro de montagem contendo 1,5 ug / ml de DAPI (4,6-diamidino-2-fenilindol) para contracoloração de DNA total. Lamelas são colocados lado a célula para baixo em uma queda de 10-15 ul de meio de montagem de um slide e selado com unha polonês. As lâminas são mantidos durante meses a -20 ° C. </li></ol> 6. Microscopia 3D e Análise <ol><li> Imagens em 3D podem ser adquiridos com diversos sistemas de microscópio. Nas nossas experiências, secções ópticas separadas por 0,3 pm são recolhidas num AOBS SP5 Leica sistema confocal (acústico-óptico divisor de feixe). Note-se que a separação entre optical aviões pode ser ajustada dependendo do tipo de análise. </li><li> Pilhas de imagens em 3D podem ser analisados ​​utilizando um software diferente e ferramentas. Usamos o software Image J para avaliar medidas de distância entre loci de interesse e analisar diferentes tipos de associação dos locos de interesse com sub-compartimentos nucleares ou proteínas. </li><li> Todas as análises estatísticas foram realizadas para comparar dois (ou mais) diferentes condições biológicas com pairwise avaliação (s) de significância: comparação entre diferentes tipos de células ou estágios, a comparação entre os diferentes loci de interesse. Em todos os testes estatísticos, valores de P ≤ 5.00E-2 (a = 0,05) estão a ser tomadas significativa (1.00E-2 ≤ P ≤ 5.00E-2 * significativo; 1.00E-3 ≤ P ≤ 1.00E-2 * * muito significativa, P &lt;1.00E-3 *** altamente significativo). </li></ol> A análise estatística das distribuições inteiras de distâncias entre dois alelos. Nós c primeiroonstruct curvas de freqüência acumulada em toda a faixa de distâncias entre dois alleles.To avaliar a significância das diferenças nas distribuições de empíricos inter-alélicas distâncias usamos o teste não paramétrico de duas amostras de Kolmogorov-Smirnov (KS) 13,14. A análise estatística da associação estreita (ou seja, o emparelhamento) de dois alelos usando uma distância ideal de corte. Para identificar a gama de a maioria das diferenças robustas em distâncias entre dois alelos ou loci, usamos uma série de bicaudal teste exato de Fisher 15 s. Ou seja, em cada distância medida que testa se uma condição é significativamente sobre-representados em distâncias mais curtas entre as duas amostras. A mínima na distribuição dos valores de P indica a gama de distâncias que devem ser considerados como um ponto de corte para a íntima associação (emparelhamento ie) um dos dois alelos. Posteriormente, a bicaudal teste exato de Fisher é usado tó avaliar a importância do emparelhamento de dois alelos identificados no valor de corte 15. Correcções de teste são aplicados a contabilizar o número total de testes de Fisher realizada 16. A análise estatística da associação entre o locus de interesse com sub-compartimentos nucleares ou proteínas. O teste de duas caudas exacto de Fisher-é utilizado para analisar a importância da associação do locus de interesse com compartimentos diferentes ou proteínas 15.

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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. 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Os autores declaram que não têm interesses conflitantes financeiros.

As técnicas descritas acima foram usadas no nosso laboratório para analisar a regulação da recombinação V (D) J de imunoglobulina e Tcra / d loci no desenvolvimento de linfócitos 30,31. Estamos confiantes de que esta técnica pode ser adaptada para a detecção de várias proteínas nucleares, compartimentos nucleares e locos, em diferentes tipos de células. Modificações do protocolo pode ser necessário, e, neste caso, os passos principais para se concentrar são as seguintes. Em ...

Estabelecimento de mecanismos epigenéticos gatilho e herança de desenvolvimento e de células do tipo perfis específicos de transcrição. Em um nível, este envolve a modulação da embalagem da cromatina, que define as regiões genômicas ativas ou em silêncio. Numa escala maior, a organização 3D global do genoma e arquitectura nuclear também desempenhar um papel no controlo da transcrição de padrões. Assim, o esvaziamento desses recursos epigenômico é essencial para um entendimento completo de como os genes são regulados 6-11. FISH DNA combinado imunofluorescência e 3D fornecem uma oportunidade única para complementar análises moleculares e bioquímicos, avaliando interacções específicas / associações de sequências de ADN e / ou proteínas dentro do núcleo. Além disso, enquanto do genoma técnicas de alto rendimento, como cromatina imunoprecipitação (CHIP-seq) ou conformação cromossomo captura juntamente com o seqüenciamento de profundidade (4C-seq, 5C, Hi-C) fornecer dat mundialuma população de células em 12, técnicas de imunofluorescência / ADN FISH permitir análises ao nível da célula individual. Aqui nós descrevemos um protocolo para a detecção simultânea de modificações de histonas por seqüências de DNA por imunofluorescência e FISH DNA seguido por microscopia 3D e análises (3D imuno-FISH). A vantagem deste protocolo é a visualização conjunta de DNA e preservação de estruturas de proteínas. A nossa experiência neste campo nos permitiu melhorar e simplificar os protocolos existentes. Apesar de termos utilizados neste protocolo para a detecção de ADN de cadeia dupla em intervalos linfócitos submetidos a recombinação, este método pode ser aplicado a outras proteínas e de outros tipos de células.

<table border="1"><tbody><tr><td> Nome do reagente / Material </td><td> Companhia </td><td> Número de Catálogo </td><td> Comentários </td></tr><tr><td> H 2 O </td><td> Pescador </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 a C-11401 </td><td></td></tr><tr><td> Cy3 ou Cy5 dUTP </td><td> Pescador </td><td> N º 45-001-xxx </td><td></td></tr><tr><td> DNase I </td><td> Roche </td><td> # 04536282001 </td><td></td></tr><tr><td> DNA Pol I </td><td> Biolabs </td><td> # M0209 </td><td></td></tr><tr><td> 0,025 mM filtros </td><td> Millipore </td><td> # VSWP02500 </td><td></td></tr><tr><td> Cot-1 DNA 1 mg / ml </td><td> Invitrogen </td><td> # 18440 </td><td></td></tr><tr><td> Hybloc ADN de 1 mg / ml </td><td> Genética Aplicada </td><td> # MHB </td><td></td></tr><tr><td> Esperma de salmão </td><td> Sigma </td><td> # D1626 </td><td> pó a serem ressuspensas a 10 mg / ml em H2O </td></tr><tr><td> NaAc (Acetato de sódio, pH 5,2, solução tampão) </td><td> Sigma </td><td> # S7899 </td><td></td></tr><tr><td> Ficoll 400 (Mol. Biol grau) </td><td> Pescador </td><td> # 525 </td><td></td></tr><tr><td> Polivinilpirrolidona (Mol Biol grau) </td><td> Pescador </td><td> # BP431 </td><td></td></tr><tr><td> Dextran pó de sulfato </td><td> Sigma </td><td> # D8906 </td><td></td></tr><tr><td> SSPE (Solução salina-fosfato de sódio-EDTA) solução 20x </td&gt; <td> Pescador </td><td> # BP1328 </td><td></td></tr><tr><td> Formamida </td><td> Pescador </td><td> # BP227 </td><td></td></tr><tr><td> Lamelas </td><td> Pescador </td><td> º 12-548-B </td><td></td></tr><tr><td> Slides </td><td> Pescador </td><td> # 12-550 </td><td></td></tr><tr><td> Placas de 6 poços </td><td> Pescador </td><td> # 0720080 </td><td></td></tr><tr><td> PBS, 10x </td><td> Pescador </td><td> # MT-46-013-CM </td><td></td></tr><tr><td> Poli-L-lisina solução </td><td> Sigma </td><td> # P8920 </td><td></td></tr><tr><td> Paraformaldeído, grânulos, 95% </td><td> Sigma </td><td> # 441244 </td><td></td></tr><tr><td> Triton-X-100, Mol Biol grau </td><td> Sigma </td><td> # T8787 </td><td></td></tr><tr><td> BSA (Albumina de Soro Bovino) Fracção V </td><td> Pescador </td><td> # BP 1600 </td><td></td></tr><tr><td> Soro normal de cabra </td><td> Vector Labs </td><td> # S-1000 </td><td></td></tr><tr><td> Tween-20, Mol Biol grau </td><td> Sigma </td><td> # P9416 </td><td></td></tr><tr><td> SSC (citrato de sódio Saline) solução 20x </td><td> Pescador </td><td> # BP1325 </td><td></td></tr><tr><td> Prolongar reagente Antifade Ouro </td><td> Invitrogen </td><td> # P36930 </td><td></td></tr><tr><td> DAPI (4 &#39;,6-diamidino-2-fenilindol) </td><td> Sigma </td><td> # D9542 </td><td></td></tr><tr><td> Melhor teste de um cimento de borracha casaco </td><td> Abastecimento de lojas de arte ou de escritório </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> Tabela 1. Reagentes específicos e pequenos equipamentos. </td></tr></tbody></table>

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

Hibridização fluorescente in situ utilizando sondas de DNA em 3-dimensionalmente núcleos preservados seguido por microscopia confocal 3D (3D FISH DNA) representa a forma mais direta de visualizar a localização de locos gênicos, cromossômica sub-regiões ou territórios inteiros em células individuais. Este tipo de análise fornece insights sobre a arquitetura global do núcleo, bem como o comportamento de loci genômicos e regiões específicas dentro do espaço nuclear. Imunofluorescência, por outro lado, permite a detecção de proteínas nucleares (histonas modificados, variantes de histonas e modificadores, maquinaria de transcrição e factores nucleares, sub-compartimentos, etc.) O principal desafio na combinação FISH DNA imunofluorescência e 3D é, por um lado, para preservar o epitopo detectada pelo anticorpo, bem como a arquitectura 3D do núcleo, e, por outro lado, para permitir a penetração da sonda de ADN para detectar locos gênicos ou territórios cromossômicos 1-5. Aqui nós fornecemos um protocolo que combina visualização de modificações da cromatina com loci genômicos em núcleos preservados 3D.

DNA e imuno-FISH são utilizados no laboratório Skok para estudar as alterações na organização nuclear associadas com o processo de recombinação V (D) J de receptor de antigénio, durante loci B e T o desenvolvimento de linfócitos. As técnicas acima descritas nos permitir i) medir distâncias entre as duas extremidades de um locus (contração) ii) medir distâncias entre alelos ou loci (emparelhamento), iii) analisar os danos no DNA que ocorrem dentro loci, iv) avaliar a localização de alelos e locos em rela...

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

Aqui nós descrevemos um protocolo para a detecção simultânea de modificações de histonas por imunofluorescência e seqüências de DNA por FISH DNA seguido por microscopia 3D e análises (3D FISH imuno-DNA).

Imunofluorescência combinados e FISH DNA em 3D preservado núcleos interfásicos para estudar mudanças na organização nuclear 3D

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 visualizações.  New York University School of Medicine. Aqui nós descrevemos um protocolo para a detecção simultânea de modificações de histonas por imunofluorescência e seqüências de DNA por FISH DNA seguido por microscopia 3D e análises (3D FISH imuno-DNA).

Vídeo: 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.

Teste de sensibilidade visual para a velocidade e direção do movimento em Lagartos

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

Biologia

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.

Baseados em DNA Peixe Protocolo Identificação de Espécies

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.

Robust FISH DNA 3D utilizando sondas Diretamente rotuladas

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.

Avaliando Contribuições Espécies específicas Para o Desenvolvimento Craniofacial Utilizando Quimeras Codorniz-pato

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.

DNA-RNA combinado fluorescente<em&gt; Em situ</em&gt; A hibridização (FISH) para estudar inativação do cromossomo X em Diferenciada Feminino Rato Células-Tronco Embrionárias

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.

Medindo danos DNA e reparar no mouse Esplenócitos Depois crônica<em&gt; In Vivo</em&gt; A exposição a doses muito baixas de beta e gama-radiação

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.

O isolamento de regiões específicas genômico e identificação de moléculas pela Associated 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

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.

Métodos de estudo mudanças na agregação de proteínas inerente com a idade em 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

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.

Imunofluorescência sequencial e imuno-histoquímica em embriões de zebrafish Crioseccionados

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.