Name: Um Protocolo Simplificado para Microscopia de Localização de Molécula Única em Núcleos de Arabidopsis (Vídeo) | JoVE Journal
Uploaded: 2026-05-08T14:05:00.000+00:00
Duration: 9 min 54 s
Description: Aprenda microscopia de localização de molécula única (dSTORM) para visualizar a organização da cromatina em núcleos de Arabidopsis thaliana. Este protocolo simplificado melhora a pureza dos núcleos e a consistência da marcação.

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<ol>
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<table><tbody><tr><td>Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody</td><td>Abcam</td><td>ab5095</td><td>Primary antibody used at 1/200 dilution in immunostaining buffer to visualize the active form of the polymerase (Polyclonal antibody raised in rabbit )</td></tr><tr><td>Acquisition software</td><td>Abbelight</td><td>NEO LiveImaging v2.18</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A7906&nbsp;&nbsp;</td><td></td></tr><tr><td>Catalase</td><td>Sigma-Aldrich</td><td>C40</td><td></td></tr><tr><td>Certified Low Melting Agarose</td><td>Bio-Rad</td><td>1613111</td><td></td></tr><tr><td>Coverslips thickness 1.5H round</td><td>Marienfeld</td><td>117640</td><td>&Oslash;: 24 mm</td></tr><tr><td>D-(+)-Glucose, anhydrous, 99%</td><td>Thermo Scientific</td><td>A16828-36&nbsp;</td><td></td></tr><tr><td>Dichroic mirror</td><td>Semrock</td><td>Di03-R405/488/561/635-t1</td><td>Dichroic mirror for excitation/emission separation</td></tr><tr><td>Dulbecco&rsquo;s Phosphate Buffered Saline 10&acute;</td><td>biowest</td><td>X0515 - 500&nbsp;</td><td>Work in sterile conditions</td></tr><tr><td>Epredia Ultra Disposable Microtome Blades</td><td>FisherScientific</td><td>12191830</td><td>Low profile, single use</td></tr><tr><td>Falcon 6-well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate, with Lid</td><td>&nbsp;Falcon</td><td>353046</td><td></td></tr><tr><td>Formaldehyde solution 37%</td><td>Carl Roth</td><td>7398</td><td>Work under the fume hood</td></tr><tr><td>Glucose oxidase</td><td>Sigma-Aldrich</td><td>G2133</td><td></td></tr><tr><td>Glycerol</td><td>VWR</td><td>24388.295</td><td></td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647</td><td>Thermo Scientific</td><td>A32733TR</td><td>Used at 1/200 as secondary antibody coupled to AF647</td></tr><tr><td>Hoechst 33258 solution</td><td>Sigma-Aldrich</td><td>94403</td><td>Used at 1/500 to counterstain the DNA, added to the agarose pad</td></tr><tr><td>Hydrochloric Acid Solution</td><td>Chem-Lab</td><td>CL05.0311.1000&nbsp;</td><td></td></tr><tr><td>JF549-Hoechst</td><td>Lavis lab and Open Chemistry team (Janelia)</td><td>JF549-Hoechst</td><td>Used to counterstain DNA, added to the agarose pad at 1.5 nM final concentration</td></tr><tr><td>KIMBLE Dounce tissue grinder set</td><td>Sigma-Aldrich</td><td>D8938</td><td>2 mL tube with both large (0.0030-0.0050 inch) and small (0.0005-0.0025 inch) clearance pestles</td></tr><tr><td>Lasers unit</td><td>Oxxius</td><td>L6Cc</td><td>Equipped with six lasers: 405 nm - 100 mW, 488 nm - 200 mW, 532 nm - 500 mW, 561 nm - 300 mW, 640 nm - 500 mW and 730 nm - 30 mW</td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Carl Roth</td><td>HN03</td><td></td></tr><tr><td>Mercaptoethylamine (MEA)</td><td>Sigma-Aldrich</td><td>30070</td><td></td></tr><tr><td>MOPS sodium salt 98%</td><td>Fisher Scientific SAS</td><td>352590010</td><td></td></tr><tr><td>Multiband emission filter</td><td>Semrock</td><td>FF01-446/523/600/677</td><td>Multiband emission filtration to block laser scatter light</td></tr><tr><td>Nanodiamonds</td><td>Adamas Nanotechnologies</td><td>NDNV100nmHiWGA2ml</td><td>Stock 1 mg/mL</td></tr><tr><td>ORCA-Fusion Digital CMOS camera</td><td>Hamamatsu</td><td>C14440-20UP&nbsp;</td><td></td></tr><tr><td>Petri dish, 100/20 mm, PS, clear, with vents, sterile</td><td>Greiner Bio-One</td><td>664161</td><td>Used to grow plants on 1/2 MS medium</td></tr><tr><td>Petri dish, square, PS, clear, 120/120/17 mm, sterile</td><td>Greiner Bio-One</td><td>688161</td><td>Used when chopping the plant seedlings</td></tr><tr><td>pluriStrainer Mini cell strainer</td><td>pluriSelect</td><td>43-10030-50</td><td>Mesh size 30 &micro;m, suitable for 1.5 mL Eppendorf tube</td></tr><tr><td>Potassium Chloride</td><td>Sigma-Aldrich</td><td>P9333</td><td></td></tr><tr><td>Single band emission filter</td><td>Semrock</td><td>FF01-698/70</td><td>Specific emission filter for AF647 channel</td></tr><tr><td>Single band emission filter</td><td>Chroma</td><td>ET600/50m</td><td>Specific emission filter for JF549 channel</td></tr><tr><td>Sodium chloride (99,5%)&nbsp;</td><td>Euromedex</td><td>1112-A</td><td></td></tr><tr><td>Star-Frost slides 76 x 26 mm</td><td>Knittel</td><td>&nbsp;VS112711FKB.01</td><td></td></tr><tr><td>Sterile syringe filter&oslash; 33 mm porosity 0.2 &micro;m Luer Lock cone</td><td>ClearLine</td><td>146560</td><td></td></tr><tr><td>Super-resolution system</td><td>Abbelight</td><td>SAFe360</td><td>Olympus IX83 equipped with 100x NA1.5 oil-immersion objective</td></tr><tr><td>Tri-Sodium Citrate 2H2O Gen-Apex Biomole</td><td>Prolabo</td><td>PRO-33615.268</td><td></td></tr><tr><td>Tris Base Molecular Biology Grade</td><td>Promega</td><td>H5135</td><td></td></tr><tr><td>Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)</td><td>Thermo Scientific</td><td>20491</td><td></td></tr><tr><td>Triton X-100</td><td>Euromedex</td><td>2000-B</td><td></td></tr><tr><td>Tween 20</td><td>Euromedex</td><td>2001-B</td><td></td></tr><tr><td>Twinsil Speed</td><td>Picodent</td><td>13001002</td><td>Silicone sealant</td></tr><tr><td>Ultraviolet Ozone cleaning system UVOCS oven</td><td>UVOCS Inc.</td><td>T10X10</td><td>20 min exposure for coverslip cleaning&nbsp;</td></tr><tr><td>Vacuum pump</td><td>Vacuubrand</td><td>VP 100C</td><td></td></tr><tr><td>Heraeus Megafuge 16R Centrifuge</td><td>Thermo Scientific</td><td></td><td>Rotor TX-400 75003629. Centrifugation performed with Eppendorf tubes.</td></tr></tbody></table>

um protocolo simplificado para microscopia de localização de molécula Única em núcleos de arabidopsis

A Streamlined Protocol for Single-Molecule Localization Microscopy in Arabidopsis Nuclei

Um Protocolo Simplificado para Microscopia de Localização de Molécula Única em Núcleos de Arabidopsis

While confocal fluorescence microscopy has provided valuable insights into chromatin organization in plant nuclei, its diffraction-limited resolution ...

a-streamlined-protocol-for-single-molecule-localization-microscopy

Research

JoVE Journal

Biology

Biologia

Análise de célula específico<em&gt; Arabidopsis</em&gt; Folhas utilizando ordenação células activadas por fluorescência

Nós fornecemos aqui um protocolo eficiente e confiável para a imunocoloração, hibridação in situ fluorescente, a coloração do DNA seguido de quantitativo, imagem de alta resolução em Arabidopsis thaliana óvulos inteiros de montagem. Este método foi utilizado com sucesso para analisar as modificações da cromatina e arquitectura nuclear.

Um método eficiente para Quantitativa, Análise de células Único de cromatina Modificação e Arquitetura Nuclear em Todo-mount óvulos em<em&gt; Arabidopsis</em

Temos desenvolvido um novo método para expressar co várias proteínas da fusão fluorescente quimérico em plantas para superar as dificuldades dos métodos convencionais. Ele tira vantagem do uso de um plasmídeo de expressão única que contém várias proteínas funcionalmente independentes expressando cassettes para alcançar expressão co de proteína.

Co de expressão de várias proteínas da fusão fluorescente quimérico de forma eficiente em plantas

Nós relatamos um método simples e versátil para executar ao vivo-imagem fluorescente de Arabidopsis thaliana folhas durante um período prolongado de tempo. Nós usamos uma Arabidopsis de plantas transgênicas expressando um gene repórter fluorescente sob o controle de um promotor de imunidade-relacionados como um exemplo para ganhar spatiotemporal compreensão das respostas imunes de planta.

Um método versátil para a montagem de Arabidopsis deixa para Intravital imagem de lapso de tempo

a microscopia da localização da único-molécula 3D é utilizada para sondar as posições espaciais e as trajetórias do movimento de proteínas cDNAs etiquetadas em pilhas bacterianas vivas. O protocolo de análise experimental e de dados descrito neste documento determina os comportamentos diffusivos prevalentes de proteínas citosómicas baseadas em trajetórias unimoléculas agrupadas.

Microscopia de seguimento da único-molécula-uma ferramenta para determinar os Estados diffusive de moléculas cytosolic

Aqui descrevemos um protocolo para imagens de células vivas do citoesqueleto de microtúbulos cortical no meristem apical de tiro e monitorando sua resposta às mudanças nas forças físicas.

Imagem celular ao vivo do citoesqueleto microtubulo e manipulação micromecânica da Arabidopsis Shoot Apical Meristem

O movimento cromossômico durante a prófase meiótica é um fenômeno pouco compreendido que é fundamental para a formação de esporos. Este protocolo descreve um método para adquirir, analisar e quantificar os movimentos do centrômero durante a prófase meiótica em meiócitos masculinos de Arabidopsis thaliana .

Caracterização por imagem ao vivo dos movimentos do centrômero durante a prófase meiótica masculina em Arabidopsis thaliana

Apresentamos um protocolo de microscopia de localização de molécula única em três cores (SMLM) que permite o mapeamento reprodutível de eucromatina, heterocromatina e RNAP II para análise espacial. Esse protocolo permite a marcação eficiente de multicor em ambientes nucleares densos, incluindo alvos associados à cromatina, possibilitando detecção simultânea e confiável.

Um Protocolo de Microscopia de Localização de Molécula Única Multirótulo para Investigação da Cromatina em Ambiente Nuclear Denso

Esse protocolo de imunolocalização nos meiócitos machos do milho mantém a integridade tridimensional das células durante a meiose. Ele permite o rastreamento dinâmico da localização intracelular de até três anticorpos independentes em material preservado fixado com paraformaldeído, além do DAPI, que tinge o DNA.

Imunolocalização Tridimensional em Meiocitos de Milho

Esse processo utiliza microscopia confocal em conjunto com um núcleo e linha marcadora de cromatina marcados fluorescentemente em Arabidopsis thaliana para registrar simultaneamente a dinâmica do núcleo e da cromatina nas raízes de Arabidopsis e investigar mudanças nessas dinâmicas em resposta a sinais de desenvolvimento e ambientais.

Imagem quantitativa de células vivas da forma nuclear e dinâmica da cromatina durante o desenvolvimento e estresse ambiental na raiz de Arabidopsis thaliana

Aprenda microscopia de localização de molécula única (dSTORM) para visualizar a organização da cromatina em núcleos de Arabidopsis thaliana. Este protocolo simplificado melhora a pureza dos núcleos e a consistência da marcação.

Um Protocolo Simplificado para Microscopia de Localização de Molécula Única em Núcleos de Arabidopsis

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