Vorremmo ringraziare i membri del laboratorio Skok, soprattutto Susannah Hewitt, per le discussioni e commenti. Questo lavoro è supportato dal National Institute of sovvenzioni Salute R01GM086852, RC1CA145746 (JAS). JAS è un Leukemia &amp; Lymphoma Society studioso. JC è un Fellow Irvington Istituto del Cancer Research Institute. MM è supportato da una formazione National Science Foundation Graduate Concessione Integrativa e tirocinio di ricerca (NSF IGERT 0333389).

1. DNA Etichettatura Sonda con Fluorofori: Nick Traduzione (~ 6 ore) <ol><li> Pulito BAC DNA (preparato da maxi-prep) o plasmidi o prodotti PCR, tutti risospeso in H 2 O, possono essere utilizzati per l&#39;etichettatura. Noti che per un segnale FISH robusta, le sonde devono estendersi almeno 10 kb. </li><li> Incubare DNA in RNasi A per 30 min a 37 ° C (Tutti i reagenti sono elencati nella Tabella 1). </li><li> Incubare la reazione traduzione nick per 2 ore a 16 ° C (vedi tabelle 2 e 3). Metodi alternativi di etichettatura diretta può essere utilizzato (esempio: Tag FISH, Invitrogen). </li><li> Inattivare la reazione per un ora a -80 ° C o per una notte a -20 ° C. </li><li> Determinare le dimensioni della sonda su un gel di agarosio al 2%. Lo striscio dovrebbe essere compreso tra 100 e 1.000 bp, con la maggioranza di circa 300-500 bp (Si noti che la macchia di Cy5-sonde non è visibile). Se il DNA non è digerito abbastanza, più DNA Pol I eDNasi I può essere aggiunto alla reazione ed incubata per due ore più a 16 ° C. </li><li> Purificare sonde su filtri 0,025 mm in due bicchieri litro riempite con H 2 0 per due ore al buio. </li><li> Aggiungere fattori bloccanti alle sonde come segue: 10 mg di DNA Cot-1, 10 ug di DNA Hybloc e 100 pg di sperma di salmone per 10 pg di sonde di DNA. DNA sonde Conservare a -20 ° C. </li></ol> 2. Sonda Precipitazioni / Denaturazione / Pre-ricottura (ore 3-4) <ol><li> Tra 0,3 mg e 1 mg di test sul DNA viene utilizzato per coprioggetto. Sonde mescolare con 0,1 volume di 3M NaAc (pH 5,2) e 2,5 volumi di etanolo al 100%, incubare per un ora a -80 ° C o per una notte a -20 ° C e centrifugare a 13.000 rpm a 4 ° C per 30 min. Si noti che la quantità di sonda di DNA può essere regolata a seconda della qualità del DNA nucleare e la regione di ibridazione. Metodi alternativi di etichettatura diretta può consentire l&#39;utilizzo di ridotte quantità disonda. Per il rilevamento di segnali multipli FISH DNA su una diapositiva, diverse sonde può essere precipitato insieme (tranne le sonde che non dovrebbero essere pre-cotto, come sequenze ripetute). Sonde per diversi esperimenti eseguiti contemporaneamente può anche essere precipitato insieme. Pellet dovrebbe essere leggermente colorata secondo i fluorofori utilizzati (se piccole quantità di sonde vengono precipitati, il pellet può non essere visibile). </li><li> Pellet secco e risospendere in 15 microlitri tampone di ibridazione (cfr. tabella 4) per coprioggetto con agitazione (con un Thermomixer Eppendorf) al buio a 37-45 ° C (per circa 20 min). </li><li> Sonde Denaturare per 5 minuti a 75-95 ° C. </li><li> Pre-ricottura sonde a 37 ° C per 30-60 min. Lunghezza del pre-ricottura può essere regolata a seconda della complessità e qualità delle sonde. </li><li> Applicare i coprioggetti sonde per l&#39;ibridazione durante la notte (cfr. sezione 3). </li></ol> 3. 3-Dimensionale FISH DNA - Primo Giorno (~ 3 ore) <ol><li> Cellule non aderenti sono concentrate in un piccolo volume di PBS 1x (~ 2x10 5 cellule in 5-10 microlitri 1x PBS per vetrino) e scaricati sul centro di un poli-L-lisina coprioggetto rivestiti. Cellule aderenti dovrebbero essere coltivate su vetrini per almeno 24 ore (confluenza ~ 70%) (vetrini possono essere rivestiti con gelatina 0,1%). Quadrati 18x18 a 24-24 coprioggetti mm sono poste in piastre da 6 pozzetti, e tutto il protocollo, due ml di ciascuna soluzione viene usato per pozzetto (in alternativa rotonde coprioggetto 1,5 millimetri può essere posto in piastre 12/24-well). </li><li> Fissare cellule in paraformaldeide al 2% / PBS 1x, pH 7-7,4, per 10 minuti a RT (4% PFA scorte possono essere aliquotati, conservati a -20 ° C). Paraformaldeide possono essere acquistati in soluzione (16%), o soluzioni di riserva del 4% può essere preparato da polvere / granuli. Paraformaldeide in polvere viene dissolto in PBS 1x a&gt; 60 ° C (pH può essere portato a 8-9 per facilitare la dissoluzione), raffreddato in ghiaccio, regolareed a pH 7-7,4, filtrata, aliquotare falchi e conservati per settimane a -20 ° C. </li><li> Dopo tre lavaggi in PBS 1x, permeabilizzare le cellule in ghiaccio-freddo 0,4% Triton-X-100 / 1x PBS per 5 minuti in ghiaccio. Lunghezza di permeabilizzazione può essere regolata a seconda del tipo cellulare. NB: Se FISH DNA viene combinato con immunofluorescenza, l&#39;immunocolorazione deve essere eseguita in questa fase (vedi sezione 4). </li><li> Dopo tre lavaggi in PBS 1x, incubare le cellule con 0,1 mg / microlitri RNasi A in PBS 1x per un ora a 37 ° C. Coprioggetti sono posti cellula-lato giù su un carico di 100 pl di soluzione su parafilm o un vetrino in una camera umida. Coprioggetto vengono poi accuratamente rimosso con una pinza. Se si incontra una resistenza, coprioggetto deve essere allagato con PBS 1x per evitare di danneggiare le cellule. </li><li> Dopo tre lavaggi in PBS 1x, permeabilizzare le cellule in ghiaccio-freddo 0,7% Triton-X-100 / HCl 0,1 M per 10 min in ghiaccio. </li><li> Dopo tre lavaggi in 1x PBS, le cellule denaturate in HCl 1,9 M per 30 minuti a RT. Alternativamente, le cellule possono essere denaturati in 50% di formammide / 2x SSC a 75-80 ° C per almeno 30 min. Notare che la lunghezza ibridazione (e temperatura) può essere ottimizzato a seconda del tipo cellulare. </li><li> Dopo tre lavaggi in ghiacciata 1x PBS, le cellule ibridare con sonde notte a 37 ° C in una camera oscura e umida. Coprioggetto sono posti cellulare rivolto verso il basso su un calo del 15 microlitri di sonda su un vetrino, e sigillato con mastice. </li></ol> 4. 3-dimensionale Immuno-FISH - Primo Giorno (~ 6-7 ore) <ol><li> Per combinato immunofluorescenza / DNA FISH, immuno-colorazione deve essere eseguita dopo la permeabilizzazione nello 0,4% Triton-X-100 (punto 3.3). </li><li> Incubare le cellule in soluzione di blocco per 30 minuti a RT (2,5% di sieroalbumina bovina (BSA), 10% siero di capra normale, 0,1% Tween-20). La soluzione bloccante è filtrata, aliquotare Eppendorfs 1,5 ml e conservati per mesi a -20 ° C. </li><li> Incubare le cellule con l&#39;anticorpo primario sollevata contro la proteina o la modificazione degli istoni di interesse diluito in soluzione di bloccaggio, per un ora a RT in una camera umida. Qui vi mostriamo l&#39;esempio di un anticorpo contro fosforilati della serina-139 di H2AX (γ-H2AX, Millipore: vedi Tabella 5 per i dettagli). Coprioggetti sono posti cellula-lato giù su un carico di 100 pl di soluzione su parafilm o un vetrino in una camera umida. Coprioggetti sono poi accuratamente rimosso con una pinza (inondato con PBS 1x se necessario). Notare che la diluizione e la lunghezza di incubazione può essere regolata a seconda della qualità del anticorpo e cellule-tipi. </li><li> Lavare le cellule in 0,2% BSA / 0,1% Tween-20 / PBS 1x, tre volte 5 min a temperatura ambiente con agitazione. </li><li> Incubare le cellule con il fluoroforo appropriato anticorpo secondario coniugato diluito in soluzione di blocco per un ora a RT in una camera oscura e umida. Qui mostriamo un esempio di rilevamento con unsecondario di capra anti-topo (Alexa Fluor 488 o 555, Invitrogen, vedere la Tabella 5 per i dettagli). Anticorpi secondari coniugati aptene-(biotina, digossigenina, etc) potrebbe anche essere usato. In questo caso, un ulteriore passaggio per il rilevamento appropriato (streptavidina, anti-dig, ecc) può essere eseguita prima o dopo over-notte ibridazione DNA-FISH. </li><li> Risciacquare le cellule in 0,1% di Tween-20 / PBS 1x, tre volte 5 min a temperatura ambiente con agitazione al buio. </li><li> Post-fix cellule nel 2% paraformaldeide / 1x PBS per 10 min a temperatura ambiente al buio. </li><li> Continua FISH DNA come sopra da incubazione a RNasi A (passo 3.4). </li></ol> 5. 3-dimensionale DNA FISH / Immuno-FISH - Secondo giorno (circa 2 ore) <ol><li> Rimuovere con cautela cemento di gomma, vetrini alluvione con 2x SSC, rimuovere con cautela con una pinza e metterli in pozzetti contenenti soluzione di lavaggio. </li><li> Lavare le cellule in 2x SSC per 30 min a 37 ° C al buio con agitazione.</li><li> Lavare le cellule in 2x SSC per 30 minuti a RT al buio con agitazione. </li><li> Lavare le cellule in 1x SSC per 30 minuti a RT al buio con agitazione noti che in caso di denaturazione con formammide, lavaggi deve essere sostituito dal seguente:. 3 lavaggi a 50% formammide lavaggi / 2x SSC e 3 in 2x SSC, 5 min ciascuna, a 37 ° C. </li><li> Montare cellule in mezzo prolungare oro montaggio contenente 1,5 mg / ml DAPI (4,6-Diamidino-2-fenilindolo) per controcolorazione di DNA totale. Coprioggetto sono posti cellulare rivolto verso il basso su un calo di 10-15 ml di mezzo di montaggio su un vetrino e sigillato con smalto. I vetrini sono conservati per mesi a -20 ° C. </li></ol> 6. Microscopia e analisi 3D <ol><li> Le immagini 3D possono essere acquisite con sistemi microscopio diversi. Nei nostri esperimenti, sezioni ottiche separate di 0,3 micron vengono raccolti su un sistema Leica SP5 AOBS confocale (acusto-ottico Beam Splitter). Notare che la distanza tra ottical piani possono essere regolati a seconda del tipo di analisi. </li><li> Stack di immagini 3D possono essere analizzati utilizzando diversi software e strumenti. Usiamo il software J immagine per valutare le misure di distanza tra i loci di interesse e di analizzare i diversi tipi di associazione dei loci di interesse con sub-nucleare compartimenti o proteine. </li><li> Tutte le analisi statistiche sono effettuate per confrontare due (o più) diverse condizioni biologiche con coppie di valutazione (s) di importanza: confronto tra diversi tipi di cellule o fasi, il confronto tra i diversi loci di interesse. In tutti i test statistici, P-value ≤ 5.00E-2 (a = 0.05) sono presi per essere significativo (1,00 E-2 ≤ P ≤ 5.00E-2 * significativo; 1.00E-3 ≤ P ≤ 1,00 E-2 * * molto significativo, p &lt;1,00 E-3 *** altamente significativo). </li></ol> Analisi statistica delle distribuzioni intero distanze tra due alleli. Abbiamo prima construct cumulativi curve di frequenza di distribuzione su tutta la gamma di distanze misurate tra i due alleles.To valutare la significatività delle differenze tra le distribuzioni empiriche di inter-alleliche distanze che utilizziamo il non parametrico due campioni di Kolmogorov-Smirnov (KS) prova 13,14. L&#39;analisi statistica di una stretta associazione (pairing cioè) di due alleli con una distanza ottimale di cut-off. Per identificare la gamma di robusti maggior parte delle differenze in termini di distanze tra due alleli o loci, si usa una serie di due code test esatto di Fisher 15 s. Vale a dire, ad ogni distanza misurata abbiamo verificare se una condizione è significativamente sovrarappresentati a brevi distanze tra i due campioni. Il minimo nella distribuzione dei valori p indica l&#39;intervallo di distanze che devono essere considerati come un cut-off per la stretta associazione (pairing cioè) dei due alleli. Successivamente il due code test esatto di Fisher è usato to valutare l&#39;importanza di accoppiamento di due alleli al identificato cut-off 15. Correzioni test multipli vengono conteggiati per il numero totale di test eseguiti Fisher 16. Analisi statistica di associazione del locus di interesse con compartimenti sub-nucleari o proteine. La due code esatto di Fisher-test viene utilizzato per analizzare il significato di associazione del locus di interesse con diversi scomparti o proteine ​​15.

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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>

Gli autori dichiarano di non avere conflitto di interessi finanziari.

Le tecniche sopra descritte sono state usate nel nostro laboratorio per analizzare la regolazione di V (D) J ricombinazione delle immunoglobuline e TCRA / d loci nello sviluppo linfociti 30,31. Siamo certi che questa tecnica può essere adattata per il rilevamento di varie proteine ​​nucleari, vani loci nucleari e, in diversi tipi di cellule-. Modifiche del protocollo può essere necessario, e in questo caso le fasi principali di concentrarsi su sono i seguenti. Primo, la lunghezza di pe...

Epigenetica meccanismi di creazione di trigger e l&#39;eredità di profili specifici di sviluppo e delle cellule di tipo trascrizionale. A un certo livello si tratta di modulazione degli imballaggi cromatina che definisce regioni genomiche attive o in silenzio. Su una scala più ampia, globale organizzazione 3D del genoma e architettura nucleare anche svolgere un ruolo nel controllo trascrizionale di modelli. Così, la dissezione di queste caratteristiche epigenomic è essenziale per una piena comprensione di come i geni sono regolati 6-11. Combinato FISH DNA immunofluorescenza e 3D offrono una possibilità unica per integrare le analisi molecolari e biochimici valutando specifiche interazioni / associazioni di sequenze di DNA e / o proteine ​​all&#39;interno del nucleo. Inoltre, mentre il genoma tecniche ad alto rendimento, come immunoprecipitazione della cromatina (ChIP-seq) o la cattura conformazione cromosoma accoppiata con sequenziamento profondo (4C-seq, 5C, Hi-C) forniscono dat globaleuna su popolazioni cellulari 12, immunofluorescenza / DNA tecniche FISH affinché le analisi a livello di singola cellula. Qui si descrive un protocollo per la rilevazione simultanea di modificazioni istoniche da sequenze di DNA di immunofluorescenza e FISH DNA seguita da microscopia e analisi 3D (3D immuno-FISH). Il vantaggio di questo protocollo è la visualizzazione combinata di DNA e conservazione delle strutture proteiche. La nostra esperienza in questo campo ci ha consentito di migliorare e semplificare i protocolli esistenti. Anche se abbiamo usato questo protocollo per rilevare il DNA a doppio filamento rompe in linfociti sottoposti ricombinazione, questo metodo può essere applicato ad altre proteine ​​e altri tipi di cella.

<table border="1"><tbody><tr><td> Nome del reagente / materiale </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> H 2 O </td><td> Pescatore </td><td> # BP2470 </td><td></td></tr><tr><td> RNasi 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 o Cy5 dUTP </td><td> Pescatore </td><td> # 45-001-xxx </td><td></td></tr><tr><td> DNasi 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 micron filtri </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 DNA 1 mg / ml </td><td> Applied Genetics </td><td> # MHB </td><td></td></tr><tr><td> Salmone sperma </td><td> Sigma </td><td> # D1626 </td><td> polvere da risospese a 10 mg / ml in H 2 O </td></tr><tr><td> NaAc (Acetato di sodio, pH 5.2, soluzione tampone) </td><td> Sigma </td><td> # S7899 </td><td></td></tr><tr><td> Ficoll 400 (Mol Biol grado) </td><td> Pescatore </td><td> # 525 </td><td></td></tr><tr><td> Polivinilpirrolidone (Mol Biol grado) </td><td> Pescatore </td><td> # BP431 </td><td></td></tr><tr><td> Destrano solfato polvere </td><td> Sigma </td><td> # D8906 </td><td></td></tr><tr><td> SSPE (Saline-Sodio Fosfato-EDTA) soluzione 20x </td&gt; <td> Pescatore </td><td> # BP1328 </td><td></td></tr><tr><td> Formammide </td><td> Pescatore </td><td> # BP227 </td><td></td></tr><tr><td> Coprioggetto </td><td> Pescatore </td><td> # 12-548-B </td><td></td></tr><tr><td> Diapositive </td><td> Pescatore </td><td> # 12-550 </td><td></td></tr><tr><td> 6-pozzetti </td><td> Pescatore </td><td> # 0720080 </td><td></td></tr><tr><td> PBS, 10x </td><td> Pescatore </td><td> # MT-46-013-CM </td><td></td></tr><tr><td> Poli-L-lisina soluzione </td><td> Sigma </td><td> # P8920 </td><td></td></tr><tr><td> Paraformaldeide, granuli, 95% </td><td> Sigma </td><td> # 441244 </td><td></td></tr><tr><td> Triton-X-100, Mol Biol grade </td><td> Sigma </td><td> # T8787 </td><td></td></tr><tr><td> BSA (albumina di siero bovino) Frazione V </td><td> Pescatore </td><td> # BP 1600 </td><td></td></tr><tr><td> Siero di capra normale </td><td> Vector Labs </td><td> # S-1000 </td><td></td></tr><tr><td> Tween-20, Mol Biol grade </td><td> Sigma </td><td> # P9416 </td><td></td></tr><tr><td> SSC (citrato di sodio Saline) 20x soluzione </td><td> Pescatore </td><td> # BP1325 </td><td></td></tr><tr><td> Prolungare reagente Oro antifade </td><td> Invitrogen </td><td> # P36930 </td><td></td></tr><tr><td> DAPI (4 &#39;,6-Diamidino-2-phenylindole) </td><td> Sigma </td><td> # D9542 </td><td></td></tr><tr><td> Miglior prova di uno strato di cemento di gomma </td><td> D&#39;arte o in ufficio di approvvigionamento negozi </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> Tabella 1. Reagenti specifici e piccole attrezzature. </td></tr></tbody></table>

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

Ibridazione fluorescente in situ con l&#39;utilizzo di sonde a DNA, il 3-dimensionale nuclei conservati seguito da 3D microscopia confocale (FISH DNA 3D) rappresenta il modo più diretto per visualizzare la posizione di loci genici, cromosomica sub-regioni o interi territori in celle singole. Questo tipo di analisi permette di comprendere meglio l&#39;architettura globale del nucleo e il comportamento dei loci genomici e regioni nello spazio nucleare. Immunofluorescenza, invece, permette la rilevazione di proteine ​​nucleari (istoni modificati, varianti istoniche e modificatori, macchinari e fattori di trascrizione nucleari, sotto-compartimenti, ecc). La sfida in combinazione FISH DNA immunofluorescenza e 3D è, da un lato di preservare l&#39;epitopo riconosciuto dagli anticorpi così come l&#39;architettura 3D del nucleo, e dall&#39;altro, per consentire la penetrazione della sonda di DNA per rilevare loci genici o territori cromosomici 1-5. Qui forniamo un protocollo che combina la visualizzazione di modificazioni della cromatina con loci genomici in 3D nuclei conservato.

DNA e immuno-FISH vengono utilizzate in laboratorio Skok di studiare le variazioni dell&#39;organizzazione nucleare associati al processo di V (D) J ricombinazione dei loci recettore per l&#39;antigene durante lo sviluppo dei linfociti B e T. Le tecniche sopra descritte ci permettono di i) misurare le distanze tra le due estremità di un locus (contrazione), ii) misurare le distanze tra alleli o loci (pairing), iii) analizzare il danno al DNA che si verificano all&#39;interno loci, iv) verificare la posizione degli alle...

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

Qui si descrive un protocollo per la rilevazione simultanea di modificazioni istoniche da sequenze di DNA di immunofluorescenza e FISH DNA seguita da microscopia e analisi 3D (3D immuno-DNA FISH).

Immunofluorescenza combinato e FISH DNA 3D conservato nuclei in interfase di studiare i cambiamenti in 3D Organizzazione nucleare

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 Views.  New York University School of Medicine. Qui si descrive un protocollo per la rilevazione simultanea di modificazioni istoniche da sequenze di DNA di immunofluorescenza e FISH DNA seguita da microscopia e analisi 3D (3D immuno-DNA FISH).

Video: 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.

Test di sensibilità visiva alla velocità e direzione del movimento in lucertole

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

Ricerca

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.

Basati sul DNA Fish Identification Specie protocollo

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.

Robusto FISH 3D DNA con sonde marcate direttamente

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.

Valutare Contributi specie-specifiche per lo sviluppo craniofacciale Uso Chimere Quaglia-anatra

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 combinato fluorescente<em&gt; In situ</em&gt; Ibridazione (FISH) per studiare inattivazione del cromosoma X nelle cellule staminali embrionali di topo differenziata Femminile

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.

Misurazione danno al DNA e ripristina in mouse Splenociti Dopo cronica<em&gt; In Vivo</em&gt; L&#39;esposizione a dosi molto basse di beta e gamma Radiazioni

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.

L&#39;isolamento di regioni genomiche specifici e l&#39;identificazione di molecole da 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.

Metodi di studio variazioni inerenti l'aggregazione proteica con l'età 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 ...

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.

Immunofluorescenza sequenziale e immunosofochimica su embrioni di pesce zebra criosezione

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.