Name: Een multilabel Single Molecule Localization Microscopieprotocol voor het Onderzoek van Chromatine in de dichte nucleaire omgeving (Video) | JoVE Journal
Uploaded: 2026-06-05T13:00:00.000+00:00
Duration: 8 min 49 s
Description: Leer een sequentiële immunolabelingmethode voor driedimensionale single molecule localization microscopie in dichte kernomgevingen. Dit protocol combineert geoptimaliseerde buffers met computationele analyse voor nanoschaal chromatineonderzoek in BJ Fibroblast, HeLa en MCF 10A cellen.

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<table><tbody><tr><td>Andor iXon Ultra 888 electron-multiplying CCD&nbsp;</td><td>Andor</td><td>DU-888U3-CSO-#BV&nbsp;</td><td>NA</td></tr><tr><td>Bovine serum albumin</td><td>Sigma Aldrich</td><td>A7030</td><td>Gebruikt in blokkerings- en wasbuffers. Bewaar BSA-gebaseerde blokkeringsbuffer niet langer dan 1 maand bij 4&deg;C</td></tr><tr><td>Changchun New Industries Optoelectronics Tech. Co., Ltd., Model MGL-FN-532 (532 nm)&nbsp;</td><td>PSU-H-LED&nbsp;</td><td>https://www.cnilaser.com/MGL-FN-532.htm</td><td>NA</td></tr><tr><td>Coherent OBIS Laser Box (405 nm, 488 nm, 532 nm, 552 nm, 637 nm)&nbsp;</td><td>Coherent</td><td>1228877&nbsp;</td><td>Lasers gecollimeerd met een gemiddeld vermogen van 3&ndash;10 kW/cm&sup3; op monster, minimaal 10.000 frames verzameld per golflengtekanaal met&nbsp;10&ndash;30&nbsp;ms&nbsp;verwervingstijd.&nbsp;</td></tr><tr><td>DABCO (1,4-Diazabicyclo-(2.2.2)-octaan)</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>Gedestilleerd water</td><td>NA</td><td>NA</td><td>Elke gedestilleerd water is prima&nbsp;</td></tr><tr><td>DTT (Dithiothreitol), 1M</td><td>Sigma</td><td>43816</td><td>NA</td></tr><tr><td>Eight well chambered cover glass</td><td>Cellvis</td><td>C8-1.5H-N</td><td>Kan elke glazen bodemplaat zijn, maar volumes moeten worden aangepast afhankelijk van het vat.</td></tr><tr><td>Goat anti Mouse AF568</td><td>Thermo Fisher</td><td>A11004</td><td>Voorraadconcentratie: 2 mg/mL Stabiliteit na etikettering: 2&ndash;3 dagen</td></tr><tr><td>Goat anti Rabbit AF647</td><td>Thermo Fisher</td><td>A21245</td><td>Voorraadconcentratie: 2 mg/mL Stabiliteit na etikettering: 4&ndash;5 dagen</td></tr><tr><td>Goat anti Rat A488</td><td>Thermo Fisher</td><td>A11006</td><td>Voorraadconcentratie: 2 mg/mL Stabiliteit na etikettering: 2&ndash;3 dagen</td></tr><tr><td>H3K27ac Primair Antilichaam</td><td>Thermo Fisher</td><td>MA5-23516</td><td>Voorraadconcentratie: 1,0 mg/mL&nbsp;</td></tr><tr><td>H3K9me3 Primair Antilichaam&nbsp;</td><td>Abcam</td><td>AB1769156</td><td>Voorraadconcentratie: 1,287 mg/mL&nbsp;</td></tr><tr><td>High Clarity Poly-Propyleen Conische buis 15 mL&nbsp;&nbsp;</td><td>Corning&nbsp;</td><td>352096</td><td>Geborekt voor fixeer- en dempingsoplossingen&nbsp;</td></tr><tr><td>High Clarity Poly-Propyleen Conische buis 50 mL</td><td>Corning</td><td>352070</td><td>Geborekt voor 40 mL werkstocks van blokkerings- en wasbuffers</td></tr><tr><td>Zoutzuur (HCl), 12M</td><td>Sigma</td><td>258148</td><td>NA</td></tr><tr><td>Nikon Eclipse Ti-E met perfect focus systeem&nbsp;</td><td>NIikon</td><td>TI-DH 611392&nbsp;</td><td>Inverteerde microscoop&nbsp;</td></tr><tr><td>Nikon SR APO TIRF, 100x vergroting, 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>Normaal geitenserum</td><td>Abcam</td><td>AB7481-1002</td><td>Gebruikt nadat de eerste label is gedaan. Moet aanwezig zijn in blokkerings- en wasbuffers</td></tr><tr><td>Paraformaldehyde 16 %&nbsp;</td><td>Electron Microscopy Sciences</td><td>15710</td><td>Gebruikt in fixeeroplossing die binnen 2 weken na het maken moet worden gebruikt. Beschermd bewaren tegen licht bij 4&deg;C</td></tr><tr><td>Fosfaat gebufferd zout (PBS) 10X</td><td>Ambion</td><td>AM9625</td><td>NA</td></tr><tr><td>Pipettips 1000 uL</td><td>SureOne</td><td>02-707-404</td><td>Elke pipettip is prima zolang deze geschikt is voor het volume</td></tr><tr><td>RNAPII-PS2</td><td>Abcam</td><td>AB252855</td><td>Voorraadconcentratie: 0,98 mg/mL</td></tr><tr><td>Natriumborohydrid</td><td>Thermo Fisher</td><td>S678-10</td><td>Gebruik vers voor dempingsbuffer elke keer opnieuw.&nbsp;</td></tr><tr><td>Natriumhydroxide(NaOH)</td><td>Thermo Fisher&nbsp;</td><td>A16037.36</td><td>NA</td></tr><tr><td>Natriumsulfiet</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>

een multilabel single molecule localization microscopieprotocol voor het onderzoek van chromatine in de dichte nucleaire omgeving

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

Een multilabel Single Molecule Localization Microscopieprotocol voor het Onderzoek van Chromatine in de dichte nucleaire omgeving

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

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Research

JoVE Journal

Bioengineering

Bio-engineering

Enkel molecuul microscopie Approach die nieuwe inzichten naar nucleaire transport.

Single-molecule beeldvorming van de nucleaire transporten

Door de combinatie van inheemse en verknopen van chromatine immunoprecipitatie met een hoge-resolutie massaspectrometrie, ChroP benadering stelt aan de samengestelde proteomics architectuur van histonmodificaties, varianten en niet-histonic eiwitten synergizing op functioneel verschillende chromatinedomeinen ontleden.

De ChroP aanpak combineert chip en massaspectrometrie te ontleden Locus-specifieke Proteomic Landschappen van Chromatin

Wij bieden hier een efficiënt en betrouwbaar protocol voor immunokleuringen, fluorescentie in situ hybridisatie, DNA kleuring gevolgd door kwantitatieve, hoge-resolutie beeldvorming in whole-mount Arabidopsis thaliana eitjes. Deze methode is succesvol gebruikt om chromatine modificaties en nucleaire architectuur te analyseren.

Een efficiënte methode voor kwantitatieve, single-cell analyse van chromatine modificatie en Nucleaire Architectuur in zijn geheel-mount Ovules in<em&gt; Arabidopsis</em

Onlangs ontdekte geoxideerde vormen van 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) en 5-carboxylcytosine (5caC) kan verschillende DNA wijzigingen met unieke functionele rollen. Hier wordt een semi-kwantitatieve workflow voor visualisatie van oxi-mCs ruimtelijke verdeling, signaal intensiteit profilering en colocalization omschreven.

Immunokleuring voor DNA wijzigingen: computationele analyse van Confocal beelden

Hier presenteren we een protocol voor Multi-Color lokalisatie van één membraaneiwitten in organellen van levende cellen. Als u wilt koppelen fluorophores, worden zelf labeling eiwitten gebruikt. Eiwitten, gelegen in verschillende membranen compartimenten van de dezelfde organel, kunnen worden gelokaliseerd met een nauwkeurigheid van ~ 18 nm.

Multi-Color lokalisatie microscopie van één membraaneiwitten in organellen van de cellen van levende zoogdieren

3D multicolor DNA FISH vertegenwoordigt een hulpmiddel om meerdere genomische loci binnen 3D bewaarde kernen te visualiseren, ondubbelzinnig definiëren d.m.v. hun wederzijdse interacties en lokalisatie binnen de nucleaire ruimte op één celniveau. Hier wordt een stap voor stap protocol beschreven voor een breed spectrum van menselijke primaire cellen.

3D Multicolor DNA FISH Tool om nucleaire architectuur in menselijke primaire cellen te bestuderen

CUT&amp;RUN en zijn varianten kunnen worden gebruikt om de eiwitbezetting op chromatine te bepalen. Dit protocol beschrijft hoe de eiwitlokalisatie op chromatine kan worden bepaald met behulp van eencellige uliCUT &amp; RUN.

Single-Cell Factor Lokalisatie op Chromatine met behulp van Ultra-Low Input Cleavage Under Targets en Release met Nuclease

Het huidige protocol beschrijft een methode voor het meten van absolute DNA-dichtheden in adherente celkernen met behulp van Voronoi-tessellatie van lokalisatiemicroscopiegegevens van één molecuul, bekend volume, genoomgrootte en celcyclusstadium.

Absolute DNA-dichtheid in celkernen in kaart brengen met behulp van lokalisatiemicroscopie met één molecuul

Dit artikel presenteert een gedetailleerde experimentele procedure voor het reconstrueren van nucleosoombevattende DNA-kettingen voor correlatieve kracht en fluorescentiemicroscopie van één molecuul. Het beschrijft verder verschillende stroomafwaartse experimenten die kunnen worden uitgevoerd om het bindingsgedrag van chromatine-interagerende eiwitten te visualiseren en veranderingen in de fysische eigenschappen van nucleosomen te analyseren.

In situ nucleosoomassemblage voor correlatieve kracht van één molecuul en fluorescentiemicroscopie

Dit protocol biedt gedetailleerde bewerkingen voor monstervoorbereiding, gegevensverzameling en analyse van DNA intrinsieke fluorescentiespectra.

Spectroscopische superresolutiebeeldvorming van DNA-moleculen met intrinsiek contrast

Dit protocol biedt een efficiënte workflow voor single-molecule localisatie microscopiebeeldvorming van geïsoleerde Arabidopsis-kernen, waarbij geoptimaliseerde isolatie, fixatie en labeling wordt gebruikt om betrouwbare nanoschaalvisualisatie van chromatine en RNA Polymerase II te bereiken. Deze benadering kan gemakkelijk worden aangepast aan andere chromatine-geassocieerde eiwitten of histonmodificaties voor beeldvorming met hoge resolutie.

Een gestroomlijnd protocol voor single-molecule localisatiemicroscopie in Arabidopsis-kernen

Leer een sequentiële immunolabelingmethode voor driedimensionale single molecule localization microscopie in dichte kernomgevingen. Dit protocol combineert geoptimaliseerde buffers met computationele analyse voor nanoschaal chromatineonderzoek in BJ Fibroblast, HeLa en MCF 10A cellen.

Een multilabel Single Molecule Localization Microscopieprotocol voor het Onderzoek van Chromatine in de dichte nucleaire omgeving (Video) | JoVE Journal

Een multilabel Single Molecule Localization Microscopieprotocol voor het Onderzoek van Chromatine in de dichte nucleaire omgeving

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