Name: 애기장대 핵에서 단일 분자 위치 현미경을 위한 간소화된 프로토콜 (동영상) | JoVE Journal
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Description: 학습하세요 애기장대 핵에서 단일 분자 위치 현미경을 위한 간소화된 프로토콜로, 고해상도 염색질 구조와 핵 조직을 시각화하는 방법입니다. 이 기법은 식물 세포 생물학 연구에 필수적인 도구를 제공합니다.

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<ol>
	<li>Misteli, T. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+the+sequence:+cellular+organization+of+genome+function.">Beyond the sequence: cellular organization of genome function.</a> Cell. 128 (4), 787-800 (2007).</li><li>Rowley, M. J., Corces, V. G. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organizational+principles+of+3D+genome+architecture.">Organizational principles of 3D genome architecture.</a> Nat Rev Genet. 19 (12), 789-800 (2018).</li><li>Fransz, P., de Jong, J. H., Lysak, M., Castiglione, M. 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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>
```

애기장대 핵에서 단일 분자 위치 현미경을 위한 간소화된 프로토콜

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

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

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Biology

생물학

의 세포 구체적인 분석<em&gt; Arabidopsis</em&gt; 형광 활성화 셀 정렬을 사용하여 잎

우리는 면역 염색, 형광 제자리 교잡, 전체 마운트 애기 장대 난자의 양적, 고해상도 이미징 다음 DNA 염색 여기를 효율적이고 신뢰할 수있는 프로토콜을 제공합니다. 이 방법은 성공적 염색질 변형 및 핵 구조를 분석하는데 사용 하였다.

전체 마운트 난자에있는 염색질 수정 및 핵 구조의 정량, 단일 세포 분석을위한 효율적인 방법<em&gt; 애기 장대</em

공동 기존 방법의 어려움을 극복 하기 위해 공장에서 여러 공상 형광 성 융해 단백질을 표현 하기 위한 새로운 방법을 개발 했습니다. 그것은 단백질 공동 식 달성 하기 위해 카세트를 표현 하는 여러 기능적으로 독립적인 단백질을 포함 하는 단일 식 플라스 미드를 사용 하 여 활용 합니다.

식물에는 효율적인 방법으로 여러 공상 형광 성 융해 단백질의 공동 식

우리는 시간의 연장된 기간 동안 형광 라이브 이미징 애기 thaliana 잎을 수행 하기 위한 간단 하 고 다재 다능 한 방법 보고. 우리는 식물 면역 반응의 spatiotemporal 이해에 대 한 예를 들어 면역 관련 발기인의 통제 형광 성 취재 원 유전자를 표현 하는 유전자 변형 애기 식물을 사용 합니다.

Intravital 시간 경과 영상에 대 한 애기 를 장착 하는 다양 한 방법을 나뭇잎합니다

3D 단일 분자 국소화 현미경 검사법은 살아있는 세균 세포에서 형광표지된 단백질의 공간 위치 및 운동 궤적을 조사하는 데 사용됩니다. 본원에 기재된 실험 및 데이터 분석 프로토콜은 풀화된 단일 분자 궤적에 기초하여 세포성 단백질의 널리 퍼진 확산 거동을 결정한다.

단일 분자 추적 현미경 검사법 - 세포화 분자의 확산 상태를 결정하는 도구

여기서 우리는 촬영 에 피질 미세 투하 세포 골격의 살아있는 세포 이미징을위한 프로토콜을 설명하고 물리적 인 힘의 변화에 대한 반응을 모니터링.

미생물의 미세 관 세포 골격및 아라비도시스 촬영 Apical 메리스템의 미세 기계적 조작의 라이브 세포 이미징

감수 분열 전기 동안의 염색체 이동은 포자 형성의 기본이 되는 잘 이해되지 않는 현상입니다. 이 프로토콜은 애기장대 탈리아나 수컷 감수 모수세포에서 감수 분열 전기 동안 중심체 움직임을 획득, 분석 및 정량화하는 방법을 설명합니다.

애기장대 의 수컷 감수 분열 전기 동안 중심체 움직임의 라이브 이미징 특성화

우리는 유크로마틴, 헤테로크로마틴, RNAP II의 재현 가능한 지도화를 가능하게 하는 3색 염색질 단분자 위치 현미경(SMLM) 염색 및 분석 프로토콜을 제시합니다. 이 프로토콜은 크로마틴 관련 표적을 포함한 고밀도 핵 환경에서 효율적인 다색 라벨링을 가능하게 하여 신뢰할 수 있는 동시 검출을 가능하게 합니다.

밀집된 핵 환경에서 크로마틴 연구를 위한 다중 표지 단일 분자 위치 현미경 프로토콜

이 수컷 옥수수 감수세포의 면역 국소화 프로토콜은 감수분열 동안 세포의 3차원적 완전성을 유지합니다. 이 방법은 보존된 파라포름알데히드 고정 물질 내에서 최대 세 개의 독립 항체의 세포 내 위치를 동적으로 추적할 수 있게 하며, DNA를 염색하는 DAPI도 포함됩니다.

옥수수 감수세포에서의 3차원 면역 위치

이 과정은 공초점 현미경과 형광 표지된 핵 및 염색질 마커 라인을 결합하여 애 기장대 뿌리의 핵과 염색질 역학을 동시에 기록하고, 발달 및 환경 신호에 반응하여 이러한 역학 변화를 조사  합니다.

애기장대 뿌리의 핵 형태와 염색질 역학의 정량적 생세포 영상화 및 발달 및 환경 스트레스 중

학습하세요 애기장대 핵에서 단일 분자 위치 현미경을 위한 간소화된 프로토콜로, 고해상도 염색질 구조와 핵 조직을 시각화하는 방법입니다. 이 기법은 식물 세포 생물학 연구에 필수적인 도구를 제공합니다.

애기장대 핵에서 단일 분자 위치 현미경을 위한 간소화된 프로토콜

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