Name: combined immunofluorescence and dna fish on 3d-preserved interphase nuclei to study changes in 3d nuclear organization
Uploaded: 2013-02-03T14:00:00
Description: 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

우리는 토론과 의견 Skok 실험실, 특히 수잔나 휴잇의 회원 감사드립니다. 이 작품은 건강 교부금의 국립 연구소 R01GM086852, RC1CA145746 (JAS)에 의해 지원됩니다. JAS는 백혈병과 림프종 사회 학자이다. JC는 암 연구소의 Irvington 연구소 연구원입니다. MM은 국립 과학 재단 (National Science Foundation) 기금 통합 대학원 교육과 연구 Traineeship (NSF IGERT 0,333,389)에 의해 지원됩니다.

1. Fluorophores있는 DNA 프로브 레이블 : 닉 번역 (~ 6 시​​간) <ol><li> 클린 BAC DNA (맥시 사립으로 작성) 또는 plasmids 또는 PCR 제품, H 2 O에 resuspended 모두, 라벨에 사용할 수 있습니다. 참고 강력한 생선 신호, 프로브 적어도 10킬로바이트에 걸쳐해야. </li><li> RNase에서 DNA를 배양 37 번 30 분에 대한 ° C (모든 시약은 표 1에 나열되어 있습니다). </li><li> 16에서 2 시간의 대화?...

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위에 설명 된 기술은 림프구에게 30,31 개발에 면역 글로불린 및 Tcra / D loci의 V (D) J 재조합의 규정을 분석하기 위해 실험실에서 사용되었습니다. 우리는이 기술이 다른 세포 유형의 다양한 핵 단백질, 핵 구획과 loci의 감지,에 적응 될 수 있다는 확신합니다. 프로토콜의 수정이 필요할 수 있습니다,이 경우에 초점을 주요 단계는 다음과 같습니다. 첫째, permeabilization의 길이는 ?...

Epigenetic 메커니즘을 트리거 설치 및 발달 및 세포 유형의 특정 전사의 프로필 상속. 한 수준에서이 활성 또는 자동 게놈 영역을 정의 염색질 포장 변조를 포함합니다. 큰 규모에서 게놈과 핵 건축의 글로벌 3D 조직은 전사 패턴의 제어 역할을한다. 따라서, 이러한 epigenomic 기능의 해부는 유전자 6-11을 규제하는 방법의 전체 이해 필수적입니다. 결합 immunofluorescence 및 3D DNA의 물고기는 특정 상호 작용 / 핵 내에서 DNA 시퀀스 및 / 또는 단백질의 연결을 평가하여 분자 및 생화학 분석을 보완 할 수있는 독특한 기회를 제공합니다. 동안 또한, 같은 염색질 immunoprecipitation (칩 seq) 또는 깊은 시퀀싱와 결합 염색체 캡쳐 형태로 게놈 전체의 높은 처리량 기술 (4C-seq, 5C는, 하이-C) 글로벌 DAT를 제공합니다세포 인구 12, immunofluorescence / DNA 물고기 기술은 단일 세포 수준에서 분석 할 수 있습니다. 여기 3D 현미경 및 분석 (3D immuno - 물고기) 다음 DNA 생선의 immunofluorescence와 DNA 시퀀스에 의한 히스톤 수정의 동시 검출을위한 프로토콜을 설명합니다. 이 프로토콜의 장점은 DNA와 단백질 구조의 보존을 결합 시각화입니다. 이 분야에 경험은 기존 프로토콜을 개선하고 단순화 할 수있게되었습니다. 우리가 재조합를 겪고 림프구에 DNA 이중 좌초 휴식을 감지하는이 프로토콜을 사용했지만,이 방법은 다른 단백질과 다른 세포 유형에 적용 할 수 있습니다.

<table border="1"><tbody><tr><td> 시약 / 재료의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> H 2 O </td><td> 어부 </td><td> # BP2470 </td><td></td></tr><tr><td> RNase </td><td> 시그마 </td><td> # R4642 </td><td></td></tr><tr><td> dNTP </td><td> 시그마 </td><td> # DNTP100 </td><td></td></tr><tr><td> 알렉사 dUTP </td><td> Invitrogen </td><td> # C11397에 대한 C-11401 </td><td></td></tr><tr><td> Cy3 또는 Cy5 dUTP </td><td> 어부 </td><td> # 45-001-XXX </td><td></td></tr><tr><td> DNase I </td><td> 로슈 </td><td> # 04536282001 </td><td></td></tr><tr><td> DNA 폴 I </td><td> Biolabs </td><td> # M0209 </td><td></td></tr><tr><td> 0.025 μm 필터 </td><td> Millipor전자 </td><td> # VSWP02500 </td><td></td></tr><tr><td> 침대-1 DNA 1 MG / ML </td><td> Invitrogen </td><td> # 18440 </td><td></td></tr><tr><td> Hybloc DNA 1 MG / ML </td><td> 응용 유전학 </td><td> # MHB </td><td></td></tr><tr><td> 연어 정자 </td><td> 시그마 </td><td> # D1626 </td><td> 분말은 H 2 O에 10 밀리그램 / ML에 resuspended 할 </td></tr><tr><td> NaAc (나트륨 아세테이트, 산도 5.2 완충 용액) </td><td> 시그마 </td><td> # S7899 </td><td></td></tr><tr><td> Ficoll 400 (몰 Biol 학년) </td><td> 어부 </td><td> # 525 </td><td></td></tr><tr><td> Polyvinylpyrrolidone (몰 Biol 학년) </td><td> 어부 </td><td> # BP431 </td><td></td></tr><tr><td> Dextran 황산 분말 </td><td> 시그마 </td><td> # D8906 </td><td></td></tr><tr><td> SSPE (식염 - 나트륨 인산-EDTA (에틸렌 다이아 민 테트라 초산)) 20x 솔루션 </td&gt; <td> 어부 </td><td> # BP1328 </td><td></td></tr><tr><td> 포름 아미드 </td><td> 어부 </td><td> # BP227 </td><td></td></tr><tr><td> Coverslips </td><td> 어부 </td><td> # 12-548-B </td><td></td></tr><tr><td> 슬라이드 </td><td> 어부 </td><td> # 12-550 </td><td></td></tr><tr><td> 6 - 잘 접시 </td><td> 어부 </td><td> # 0720080 </td><td></td></tr><tr><td> 10X PBS, </td><td> 어부 </td><td> # MT-46-013-CM </td><td></td></tr><tr><td> 폴리-L-라이신 솔루션 </td><td> 시그마 </td><td> # P8920 </td><td></td></tr><tr><td> Paraformaldehyde, prills, 95 % </td><td> 시그마 </td><td> # 441244 </td><td></td></tr><tr><td> 트리톤-X-100, 몰 Biol 학년 </td><td> 시그마 </td><td> # T8787 </td><td></td></tr><tr><td> BSA (소 혈청 알부민) 분수 V </td><td> 어부 </td><td> # BP 1600 </td><td></td></tr><tr><td> 일반 염소 혈청 </td><td> 벡터 연구소 </td><td> # S-1000 </td><td></td></tr><tr><td> 십대 초반 - 2​​0 몰 Biol 학년 </td><td> 시그마 </td><td> # P9416 </td><td></td></tr><tr><td> SSC (식염 나트륨 시트르산) 20x 솔루션 </td><td> 어부 </td><td> # BP1325 </td><td></td></tr><tr><td> 골드 antifade 시약을 연장 </td><td> Invitrogen </td><td> # P36930 </td><td></td></tr><tr><td> DAPI (4 &#39;, 6 diamidino-2-phenylindole) </td><td> 시그마 </td><td> # D9542 </td><td></td></tr><tr><td> 최고의 시험 한 코트 고무 시멘트 </td><td> 예술 또는 사무실 공급 상점 </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> 표 1. 특정 시약 및 소형 장비. </td></tr></tbody></table>

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

3D 공 촛점 현미경에 이어 3 치수 보존 핵에 DNA 프로브를 사용하여 원위치 하이브리드 화에 형광은 (3 차원 DNA 물고기) 각 셀의 하위 지역 염색체 유전자 loci의 위치, 또는 전체 지역을 시각화 할 수있는 가장 직접적인 방법을 나타냅니다. 분석이 유형의 핵의 글로벌 아키텍처뿐만 아니라 핵 공간 내의 특정 게놈의 loci와 지역의 행동에 대한 통찰력을 제공합니다. Immunofluorescence는 반면에, 핵 단백질의 검출을 (수정 histones, 히스톤 변형과 수식, 전사 기계 요소, 핵 하위 구획 등) 할 수 있습니다. immunofluorescence 및 3D DNA 물고기를 결합의 주요 과제는 핵의 3D 아키텍처뿐만 아니라 항체에 의해 검출 된 에피토프를 유지하기 위해 한 손에 있으며, 반면에, DNA 프로브의 침투가 감지 할 수 있도록 유전자 loci 또는 염색체 지역 1-5. 여기 3D 보존 핵의 게놈 loci과 염색질 수정 시각화를 결합한 프로토콜을 제공합니다.

DNA와 immuno-생선은 B와 T 림프구 개발하는 동안 항원 수용체 loci의 V (D) J 재조합의 과정과 관련된 핵 조직의 변화를 연구하기 위해 Skok 실험실에 사용됩니다. 위에 설명 된 기법은) 우리는 전 현장의 두 끝 (수축) 사이) 측정 거리에 ii) 대립 유전자 또는 loci (페어링) 사이를 측정 거리, III)가, loci에서 IV를 발생하는 DNA 손상을 분석 가능하게 대립 유전자의 위치를​​ 평가 및 하위 구획 (우리의 연구에...

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

여기 3D 현미경 및 분석 (3D immuno-DNA의 물고기)에 의해 다음 DNA 생선의 immunofluorescence와 DNA 시퀀스에 의한 히스톤 수정의 동시 검출을위한 프로토콜을 설명합니다.

3D 원자력기구의 변화를 공부하기 위해 3D 보존 된 계면 핵에 결합 Immunofluorescence와 DNA의 물고기

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

Research

JoVE Journal

Biology

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

도마뱀의 운동의 속도와 방향에 시각 민감도 테스트

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

연구

생물학

DNA-based Fish Species Identification Protocol

DNA 기반의 어종 식별 프로토콜

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

직접 레이블이 프로브를 사용하여 강력한 3D DNA 물고기

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

메추라기 오리 키메라를 사용하여 두개 안면 개발에 종 - 특정 포스트 평가

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

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

결합 된 DNA-RNA 형광<em&gt; 현장에서</em&gt; 하이브리드 화 (물고기) 차별화 된 여성 마우스 배아 줄기 세포에서 X 염색체의 불 활성화를 연구하는

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

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

만성 후 마우스 비장 세포에서 DNA 손상 및 복구를 측정<em&gt; 생체</em&gt; 베타와 감마 방사선의 매우 낮은 복용량에 노출

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high ...

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

실험 접근법은 미토콘드리아 현지화 및 핵 세포주기 키나제,는 Cdk1의 기능을 연구하는

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

특정 게놈 지역과 enChIP으로 관련 분자의 식별의 분리

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

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

꼬마 선 충 나이 연구 변화 내재 단백질 집계에 방법

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

냉동 제브라피시 배아의 순차적 면역형광 및 면역성화학

Investigation of intercellular interactions often requires discrete labeling of specific cell populations and precise protein localization. The zebrafish ...