Name: Un protocollo di microscopia a localizzazione di molecole singole multimarcatore per lindagine della cromatina nellambiente nucleare denso (Video) | JoVE Journal
Uploaded: 2026-06-05T13:00:00.000+00:00
Duration: 8 min 49 s
Description: Scopri un protocollo di microscopia a localizzazione di molecole singole multimarcatore per analizzare l'architettura della cromatina in ambienti nucleari densi utilizzando BJ Fibroblast, HeLa e MCF 10A.

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</ol>

<table><tbody><tr><td>Andor iXon Ultra 888 CCD&nbsp a moltiplicazione elettronica;</td><td>Andor</td><td>DU-888U3-CSO-#BV&nbsp;</td><td>NA</td></tr><tr><td>Albumina sierica bovina</td><td>Sigma Aldrich</td><td>A7030</td><td>Usate per bloccare e lavare i buffer. Non conservare il buffer di blocco basato su BSA per più di 1 mese a 4&deg; C</td></tr><tr><td>Changchun New Industries Optoelectronics Tech. Co., Ltd., Modello MGL-FN-532 (532 nm) e nbsp;</td><td>PSU-H-LED&nbsp;</td><td>https://www.cnilaser.com/MGL-FN-532.htm</td><td>NA</td></tr><tr><td>Scatola laser coerente OBIS (405 nm, 488 nm, 532 nm, 552 nm, 637 nm) & nbsp;</td><td>Coerente</td><td>1228877&nbsp;</td><td>I laser collimavano con 3&ndash; 10 kW/cm&sup3; Potenza media al campione, minimo 10.000 fotogrammi raccolti per canale a lunghezza d'onda con&nbsp; 10&ndash; 30&nbsp; ms&nbsp; tempo di acquisizione.&nbsp;</td></tr><tr><td>DABCO (1,4-Diazabiciclo-(2.2.2)-ottano)</td><td>Sigma</td><td>D27802</td><td>NA</td></tr><tr><td>DBPS (1X)</td><td>Thermo Fisher</td><td>14190-136</td><td>NA</td></tr><tr><td>Acqua distillata</td><td>NA</td><td>NA</td><td>Qualsiasi acqua distillata va bene.</td></tr><tr><td>DTT (Ditiotreitolo), 1M</td><td>Sigma</td><td>43816</td><td>NA</td></tr><tr><td>Vetro a copertura ben camerato</td><td>Cellvis</td><td>C8-1.5H-N</td><td>Può essere qualsiasi piastra di vetro, ma i volumi devono essere regolati a seconda del recipiente.</td></tr><tr><td>Capra anti-Mouse AF568</td><td>Thermo Fisher</td><td>A11004</td><td>Concentrazione stock: 2 mg/mL Stabilità post-etichettatura: 2&ndash; 3 giorni</td></tr><tr><td>Goat anti Rabbit AF647</td><td>Thermo Fisher</td><td>A21245</td><td>Concentrazione di stock: 2 mg/mL Stabilità post-etichettatura: 4&ndash; 5 giorni</td></tr><tr><td>Capra anti-ratto A488</td><td>Thermo Fisher</td><td>A11006</td><td>Concentrazione stock: 2 mg/mL Stabilità post-etichettatura: 2&ndash; 3 giorni</td></tr><tr><td>Anticorpo primario H3K27ac</td><td>Thermo Fisher</td><td>MA5-23516</td><td>Concentrazione di stock: 1,0 mg/mL&nbsp;</td></tr><tr><td>Anticorpi primari H3K9me3 &nbsp;</td><td>Abcam</td><td>AB1769156</td><td>Concentrazione di stock: 1,287 mg/mL&nbsp;</td></tr><tr><td>Tubo Conico di Polipropilene ad alta chiarezza 15 mL&nbsp; &nbsp;</td><td>Corning&nbsp;</td><td>352096</td><td>Utilizzato per soluzioni fissative e di tempra &nbsp;</td></tr><tr><td>Tubo conico polipropilene ad alta nitidezza 50 mL</td><td>Corning</td><td>352070</td><td>Utilizzato per scorte di lavoro da 40 mL di tamponi di blocco e lavaggio</td></tr><tr><td>Acido cloridrico (HCl), 12M</td><td>Sigma</td><td>258148</td><td>NA</td></tr><tr><td>Nikon Eclipse Ti-E con sistema di messa a fuoco perfetto &nbsp;</td><td>Nikon</td><td>TI-DH 611392&nbsp;</td><td>Microscopio rovesciato&nbsp;</td></tr><tr><td>Nikon SR APO TIRF, ingrandimento 100x, 1,49 NA&nbsp;</td><td>Nikon</td><td><a href="https://www.microscope.healthcare.nikon.com/products/optics/cfi-apochromat-tirf-series%C2%A0">https://www.microscope.healthcare.nikon.com/products/optics/cfi-apochromat-tirf-series&nbsp; </a></td><td>NA</td></tr><tr><td>Siero normale per capra</td><td>Abcam</td><td>AB7481-1002</td><td>Usato dopo che la prima etichetta è finita. Dovrebbero essere presenti nei buffer di blocco e lavaggio</td></tr><tr><td>Paraformaldehyde 16 %&nbsp;</td><td>Scienze della microscopia elettronica</td><td>15710</td><td>Usata in soluzione fissativa che dovrebbe essere consumata entro 2 settimane dalla preparazione. Proteggi dalla luce a 4°Grad; C</td></tr><tr><td>Soluzione salina tamponata con fosfato (PBS) 10X</td><td>Ambion</td><td>AM9625</td><td>NA</td></tr><tr><td>Punte per pipette 1000 uL</td><td>SureOne</td><td>02-707-404</td><td>Qualsiasi punta di pipetta va bene, purché sia così s appropriato per il volume</td></tr><tr><td>RNAPII-PS2</td><td>Abcam</td><td>AB252855</td><td>Concentrazione di stavolta: 0,98 mg/mL</td></tr><tr><td>Boridriduro di sodio</td><td>Thermo Fisher</td><td>S678-10</td><td>Usa il fresco per la tempra del buffer ogni singola volta.</td></tr><tr><td>Idrossido di sodio (NaOH)</td><td>Thermo Fisher&nbsp;</td><td>A16037.36</td><td>NA</td></tr><tr><td>Solfito di sodio</td><td>Sigma</td><td>S0505</td><td>NA</td></tr><tr><td>Triton X-100 10%</td><td>Thermo Fisher&nbsp;</td><td>28314</td><td>NA</td></tr></tbody></table>

un protocollo di microscopia a localizzazione di molecole singole multimarcatore per l'indagine della cromatina nell'ambiente nucleare denso

A Multilabel Single Molecule Localization Microscopy Protocol for Investigation of Chromatin in the Dense Nuclear Environment

Un protocollo di microscopia a localizzazione di molecole singole multimarcatore per l'indagine della cromatina nell'ambiente nucleare denso

Super-resolution microscopy has dramatically advanced our ability to interrogate biological structures beyond the diffraction limit, making it ...

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Microscopia singola molecola approch fornito intuizioni romanzo in trasporto nucleare.

Singola molecola Imaging dei trasporti nucleari

Combinando nativa e reticolazione cromatina immunoprecipitazione con alta risoluzione di Spettrometria di Massa, l'approccio ChroP permette di sezionare il composito dell'architettura proteomica delle modificazioni degli istoni, le varianti e le proteine ​​non istoniche Synergizing a domini di cromatina funzionalmente distinte.

L&#39;approccio ChroP Combina ChIP e Spettrometria di Massa a sezionare Locus-specifiche proteomica Paesaggi della cromatina

Forniamo qui un protocollo efficiente e affidabile per immunoistochimica, ibridazione in situ fluorescente, colorazione del DNA seguita da quantitativa, immagini ad alta risoluzione di tutto il montaggio di Arabidopsis thaliana ovuli. Questo metodo è stato utilizzato con successo per analizzare modificazioni della cromatina e architettura nucleare.

Un metodo efficiente per quantitativa, analisi singola cella della cromatina Cambiamento e architettura nucleare in tutto il montaggio Ovuli in<em&gt; Arabidopsis</em

Recentemente scoperto forme ossidate di 5-metilcitosina (oxi-mCs), 5-idrossimetilcitosina (5hmC), 5-formylcytosine (5fC) e 5-carboxylcytosine (5caC) può rappresentare distinte modificazioni del DNA con ruoli funzionali unici. Qui è descritto un flusso di lavoro semi-quantitativa per visualizzazione della distribuzione spaziale di oxi-mCs', profilatura di intensità di segnale e colocalizzazione.

Immunostaining per modificazioni del DNA: analisi computazionale delle immagini Confocal

Qui, presentiamo un protocollo per la localizzazione di multi-colore delle proteine di membrana singola in organelli delle cellule vive. Per allegare fluorofori, proteine self-etichettatura sono usate. Proteine, trova in scomparti di diverse membrane dell'organello stesso, possono essere localizzati con una precisione di ~ 18 nm.

Microscopia di multi-colore localizzazione delle proteine di membrana singola in organelli delle cellule di mammiferi dal vivo

IL DNA FISH multicolore 3D rappresenta uno strumento per visualizzare più loci genomici all'interno dei nuclei 3D conservati, definendo inequivocabilmente le loro interazioni reciproche e la localizzazione all'interno dello spazio nucleare a livello di singola cellula. Qui, un protocollo passo dopo passo è descritto per un ampio spettro di cellule primarie umane.

3D Multicolor DNA FISH Tool per studiare l'architettura nucleare nelle cellule primarie umane

CUT&amp;RUN e le sue varianti possono essere utilizzati per determinare l'occupazione proteica sulla cromatina. Questo protocollo descrive come determinare la localizzazione delle proteine sulla cromatina utilizzando uliCUT&amp;RUN a singola cellula.

Localizzazione del fattore a cella singola sulla cromatina utilizzando la scissione di input ultra-bassa sotto i bersagli e il rilascio utilizzando la nucleasi

Il presente protocollo descrive un metodo che misura la densità assoluta del DNA all'interno di nuclei cellulari aderenti utilizzando la tassellatura Voronoi di dati di microscopia di localizzazione di singole molecole, volume noto, dimensione del genoma e stadio del ciclo cellulare.

Mappatura della densità assoluta del DNA nei nuclei cellulari mediante microscopia a localizzazione a singola molecola

Questo articolo presenta una procedura sperimentale dettagliata per la ricostituzione di cavi di DNA contenenti nucleosomi per la forza correlativa a singola molecola e la microscopia a fluorescenza. Descrive inoltre diversi esperimenti a valle che possono essere condotti per visualizzare il comportamento di legame delle proteine che interagiscono con la cromatina e analizzare i cambiamenti nelle proprietà fisiche dei nucleosomi.

Assemblaggio di nucleosomi in situ per la microscopia a forza correlativa e fluorescenza a singola molecola

Questo protocollo fornisce operazioni dettagliate sulla preparazione del campione, raccolta dati e analisi degli spettri di fluorescenza intrinseca del DNA.

Imaging spettroscopico a super-risoluzione delle molecole di DNA utilizzando contrasto intrinseco

Questo protocollo presenta un flusso di lavoro efficiente per l'imaging microscopico di localizzazione a singola molecola di nuclei isolati di Arabidopsis, utilizzando isolamento, fissazione e marcatura ottimizzati per ottenere una visualizzazione affidabile su scala nanometrica della cromatina e della RNA Polimerasi II. Questo approccio può essere facilmente adattato ad altre proteine associate alla cromatina o modifiche agli istoni per immagini ad alta risoluzione.

Un protocollo semplificato per la microscopia di localizzazione a singola molecola nei nuclei di Arabidopsis

Scopri un protocollo di microscopia a localizzazione di molecole singole multimarcatore per analizzare l'architettura della cromatina in ambienti nucleari densi utilizzando BJ Fibroblast, HeLa e MCF 10A.

Un protocollo di microscopia a localizzazione di molecole singole multimarcatore per lindagine della cromatina nellambiente nucleare denso (Video) | JoVE Journal

Un protocollo di microscopia a localizzazione di molecole singole multimarcatore per l'indagine della cromatina nell'ambiente nucleare denso

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