Name: detection of modified forms of cytosine using sensitive immunohistochemistry
Uploaded: 2016-08-16T15:00:00.000+00:00
Duration: 7 min 13 s
Description: Watch this Scientific Journal Video about Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry at JoVE.com

We thank the Advanced Microscopy Unit (School of Life Sciences, University of Nottingham) and the Histology team of MRC Human Reproductive Sciences Unit (Edinburgh), the Institute of Neuroscience at the ULB and the FNRS for help and support. This work was supported by MRC (MR/L001047/1 to A.D.J.).

모든 동물 관련 절차는 대학의 노팅엄의 윤리 심의위원회에 따라 수행 하였다. 1. 면역 염색에 적합한 조직 준비를 선택 <ol><li> 이전 25 설명 된 바와 같이 야생형 CD1 마우스 배아 및 성인 뇌 조직의 파라핀 포함 된 섹션을 생성합니다. 4 % 포름 알데히드 (FA) 또는 방법 (25)에 대한 면역 염색을 4 % 파라 포름 알데히드 (PFA) 중 고정 뇌 조직 절편을 사용한다. 주 : 파라핀 조직 절편의 사용은 표지 된 항체에 앞서 탈 왁싱 필요하다. (2)로 처리하기 때문에 - DNA의 변성을위한 프로토콜에 사용될 4 M 염산 (HCL) 가장 항원 검색 전략 호환되지 우리 단백질 5mC 산화 유도체의 공동 검출 cryo- 또는 마이크로톰 섹션 중 하나의 사용을 권장 마커. </li></ol> 2. 탈납 파라핀 조직 절편 &lt;<html> / P&gt; <ol><li> 실행중인 클래스 II 안전 캐비닛에서, 자일 렌, 실온에서 10 분 동안 2 회 가득 코 플린 항아리에 파라핀을 내장 조직 섹션을 씻는다. 참고 : 불완전 파라핀 제거가 일치하지 않는 염색 패턴으로 이어질 수 있습니다 그것은 신선한 크실렌을 사용하는 것이 중요합니다. </li><li> 탈 왁싱 후에 빠르게 연속적으로 95, 75로 세척하여 조직 절편을 재수 화 및 RT에서 10 분마다 50 % 에탄올. </li></ol> 3. 고정 및 Cryo- 및 마이크로톰 섹션 투과성으로 <ol><li> RT에서 15 분 동안 빙냉 4 % PFA 또는 4 % FA 하나에 배치하여 재수 조직 섹션을 고정한다. </li><li> RT에서 5 분 동안 PBS의 섹션 (인산염 완충 식염수)을 세척하여 과잉 정착액을 제거합니다. </li><li> 실온에서 30 분 동안 (PBS에서 0.5 % 트리톤 X-100) PBX 가득 코 플린 항아리에 배치하여 조직 절편을 Permeabilize 하시려면. PBT (PBS에 0.​​01 % 트윈 20)에 곧 부분을 세척하여 여분의 PBX를 제거합니다. </li></ol>OXI-5mC 파생 상품에 대한 jove_title &quot;&gt; 4. 면역 염색 <ol><li> DNA의의 본 antibody에 대한 실온에서 60 분 동안 2 N 염산의 투과성이 섹션을 배치합니다. 참고 : 비록 염산의 높은 농도 (예를 들어, 4 N) 리드보다 효율적인 DNA의 변성에, 그들은 DAPI와 OXI-MCS의 공동 검출을위한 호환되지 않습니다. </li><li> HCL에 중화 RT에서 30 분 동안 10 mM 트리스 - 염산 (산도 8.5)에서 섹션을 배치합니다. 또한, 5 분 PBS의 각 섹션을 세 번 씻는다. RT에서 5 분 동안 PBT의 섹션을 품어. </li><li> 조심스럽게 부드럽게 슬라이드를 흔들어 모든 단계에서 완전히 건조 할 수있는 조직 섹션을 말도없이 조직 섹션을 둘러싼 영역에서 액체를 제거합니다. 섹션을 건드리지 않고 부분을 둘러싸 소수성 장벽 펜을 사용합니다. 주 : 소수성 장벽 펜 조직을 염색하고, 여러 섹션이 differen로 염색 할 수 있도록 필요한 항체의 양을 감소같은 슬라이드 t 항체. </li><li> 습윤 챔버에서 RT에서 1 시간 동안 차단 용액 (PBS 중 10 % 소 혈청 알부민)를 100 μL의 섹션을 인큐베이션. 참고 :이 단계를 건너 뛰는 것은 수정 된 시토신 기지를 염색의 효율성에 영향을 미치지 않을 것이다. </li><li> 마우스 단일 클론 항 5hmC 5,000 희석 및 1 : 습기 챔버에서 실온에서 1 시간 동안 차단 솔루션 토끼 다 클론 항 5caC 차 항체의 1,000 희석 1 100 ㎕의 조직 섹션을 품어. 필요한 경우 또는 4 ° C에서 하룻밤 배양을 수행합니다. 주 : 일차 항체없이 처리 섹션은 염색 절차 적합한 부정적인 컨트롤을 제공 할 수 있습니다. 12.5 일 양성 대조군으로 사용할 수 있습니다 5caC, 5fC 및 5hmC (20)이 풍부한 교미 쥐의 배아 뇌 섹션을 게시 할 수 있습니다. </li><li> 경찰에 5 분 동안 PBT 가득 코 플린 항아리에 각각 세 번 절을 세척하여 과량의 항체를 제거RT에서 린 항아리입니다. 주 : 세정액의 양을 증가 시키면 배경 얼룩을 줄일 수있다. </li><li> 과량의 PBT를 제거하고, 필요한 경우, PBT은 소수성 장벽을 약화시킬 수있는 세제를 포함하기 때문에, 소수성 장벽 펜으로 다시 섹션을 둘러싸. </li><li> 염소 항 - 토끼 HRP - 복합 항체의 400 희석 및 1 : 솔루션을 차단하는 당나귀 항 - 마우스 555 - 복합 항체의 400 희석 1을 확인합니다. </li><li> 습윤 챔버에서 실온에서 1 시간 동안 단계 4.9에서 이차 항체 혼합물을 100 ㎕의 조직 섹션을 인큐베이션. </li><li> RT에서 5 분마다 PBT로 3 회 가득 코 플린 항아리에 조직 섹션을 씻으십시오. </li><li> 실온에서 2 분 동안 tyramide 신호 증폭 버퍼에 tyramide 200 희석 : 1 100 ㎕의 조직 섹션을 배치합니다. </li><li> 즉시, PBT에서 5 분간 슬라이드를 각각 세 번 세척하여 과량 tyramide 용액을 제거한다. </li><li> 자르y를 초과 PBT를 제거하고 즉시 커버 섹션을 마운팅 중간의 드롭 (자료 목록 참조). </li><li> 부드럽게 조직 섹션에 커버 슬립을 배치하고 즉시 매니큐어로 커버 슬립을 밀봉. </li><li> 현미경 검사 전에 몇 시간 동안 4 ° C에서 조직 섹션을 저장합니다. (- 40X 배율 10) 405, 488 및 / 또는 555 nm에서 형광 현미경으로 검사합니다. </li></ol>

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The authors declare no conflict of interest.

일부 조직에서 5mC 산화 유도체, 5fC 및 5caC의보고 낮은 풍부 표준 면역 화학 프로토콜에 대한 상당한 제한을 제시 것이지만, 퍼 옥시 다제 접합 된 이차 항체의 결합은 고정 된 조직과 세포에서 이러한 시토신 수정의 검출을 허용한다 (그림 2) . 그러나 tyramide 용액 최적의 배양 시간은 신호 강도 및 tyramide 기반 신호 증폭 기간 사이에 선형 관계가 내용은 관측 tyramide 신호 증폭 키트의 각각의 배치에 대해 실험적으로 최적화되어야 알메이다 등 참조. 2012 26 . 또한, 신호 / 배경 비는 크게 초과하는 항체를 효율적으로 제거 할 수 있도록하는 코 플린 항아리에서 세척을 수행함으로써 향상 될 수있다. tyramide 용액 인큐베이션 후, 즉시 내지 D PBT 용액에 세척하여 반응을 중단하는 것이 중요ecrease 배경 염색. 이 섹션의 절차를 수행하는 동안 어느 시점에서 건조하는 것을 허용하지 않는 것이 중요합니다. DNA의 본 antibody의 효율은 37 ° C에서 본 antibody 반응을 수행함으로써 개선 될 수있다. 이 이중 가닥 DNA와 독점적으로 상호 작용으로 4 N 염산을 사용하는 대신이 있지만 N은 DAPI와 협력 염색을 허용하지 DNA의 antibody에서는 4 N 염산을 사용하여 5mC의 변형 된 형태의 염색을 향상시킨다. 이 기술은 시토신 염기 위치를 검출의 변형 형태의 강력한 반 정량적 평가를 제공 할 수 있지만, 5mC 또는 산화 유도체의 절대 레벨을 평가하기 위해 사용될 수 없다. 따라서, 우리는 다른 보완의 사용을 권장하지만 양적는 16, 19 -24 접근한다. 포유 동물 게놈에서 5mC 산화 유도체의 발견 이래, 여러 접근법은 생물학적 역할 16, 19-24을 연구하기 위해 개발되었다. 이 approa 있지만CHES가 5mC 산화 유도체의 절대 수준을 결정하는데 유용 할 수 있고, 이들은 공간적 분포 (20), (26)에 관한 정보를 제공하지 않는다. 우리는 성공적으로 상이한 세포 유형에서 5mC 산화 유도체의 공간적 분포 및 위치 파악을 매핑하기 위해 여기에 기술 된 방법을 사용한 현상과 성인 뇌 (20) 자신의 공간 분포 (20)를 공개함으로써,이 기술은 생물학적 의미와 5mC 이러한 수정 유도체는 세포 분화, 개발 및 질병을 포함하여 검출 할 수있는 다양한 생물 상황에서 5mC의 산화 파생 상품의 운명을 이해하는 데 중요 할 수 있습니다. 또, 여기서 설명하는 방법 tyrami 서로 다른 배양 시간 (염색 강도에 비례) 퍼 옥시 다제 반응의 동역학을 평가함으로써 다른 조직에서 5mC의 산화 유도체 반 정량적 평가를 제공 할 수 있습니다드 26.

DNA (5mC)에서 시토신 염기의 메틸화는 전사 1 침묵과 관련된 척추 동물의 게놈에서 발견되는 주요 후생 유전 학적 마크를 나타냅니다. 5mC 도입하고 DNA의 methyltransferases 2-5로 유지하고, 유전체 각인, X 염색체 불 활성화, 세포 분화 및 발달 3,6. 따라서, 중단 포함한 생물학적 과정의 숫자에 중요한 역할을하는 것으로 나타났다되고 5mC 게놈 패턴 질환 7, 8-11의 번호와 관련된다. 개발 및 질병에 5mC 역할을 이해하는데 진전에도 불구하고, 여전히이 마크가 개발 및 성인 조직에서 제거되는 방법을 크게 알려지지 않은 남아있다. DNA 탈 메틸화 여러 가능성 메커니즘은 최근 능동 및 수동 탈 메틸화기구 12, 13, 14, 15을 포함하여 제안되었다. 디스커버리 10-11 전위에 의해 매개 enzym 5mC 순차 산화 생성물ES (tet1 / 2 / 3)가 중간체로서 작용할 수 있는지 5 hydroxylmethylcytosine (5hmC) -5- formylcytosine (5fC) 및 DNA 16, 17, 18, ​​19 추측 자극 진핵 5- carboxylcytosine (5caC) 등 자신의 권리 (13)의 DNA 탈 메틸화 과정 또는 행위 안정적 후생 유전 학적 마크. 기본 절단 수리의 구성 요소, 티민-DNA 글리코 (TDG)가 결합하여 DNA 19 일부터 5fC과 5caC 모두 제거 할 수있는 데모, 20 활성 DNA 탈 메틸화에 수정 된 5mC 유도체의 역할을 제안하는 동안. 5fC / 5caC가 전사 조절 (29)에이 마크의 잠재적 인 참여에 RNA II의 processivity 점의 속도를 조절할 수 있음을 보여주는 최근의 증거. 인해 5mC의 산화 된 형태의 이러한 잠재적 인 생물학적 중요성 생화학 항체 기반 기술의 범위는 게놈 분포 글로벌 콘텐츠 (16), 19-24 연구에 이용되어왔다. 주어진 t의 대부분이그는 16-18, 20, 23, 25 내지 27은 서로 다른 조직에서 산화 5mC 유도체의 공간 분포를 결정하는 실험 중요해진다 장기 상이한 세포 유형으로 구성 수정 시토신 염기의 분포가 조직과 특정 세포 형인지 척추 생물학적 기능을 공개에 필요한 작업. 생화학 및 항체 기반 접근 방법의 대부분은 다른 조직과 세포 유형에 5mC의 수정 된 형태의 분포에 관한 공간 정보를 제공하지 않습니다. 대조적으로, 면역 화학 기반 기술 5mC (28) 및 산화 된 유도체 (20)의 공간적 분포와 핵 이행을 평가하는 신속한 수단을 제공 할 수있다. 즉 마우스 게놈 18 (매 10 6 시토신 3)보고 매우 낮은 5fC의 풍요 로움 (20 매 10 6 시토신에) 및 5caC 표준 면역 화학에 대한 큰 도전을 나타냅니다 말한다. 이리,우리는 강력한 제공하는 매우 민감한 면역 화학적 방법 및 포유 동물의 뇌 조직에서 시토신의 산화 된 형태의 신속 검출을 설명합니다. 신호 증폭 단계에 결합 된 퍼 옥시 다제 접합 이차 항체를 포함하여, 이러한 방법은 5fC 및 5caC의 매우 소량을 검출 문제를 회피. 또한,이 기술은 이러한 효과 후생 유전 학적 마크의 생물학적 기능을 밝히는데 다른 방법을 보완 리니지 마커 특정 사이토 공동 검출 변형 형태에 사용될 수있다.

<table><tbody><tr><td>Coplin jar</td><td>Cole-Parmer</td><td>UY-48585-30</td><td>Glass made</td></tr><tr><td>Xylene</td><td></td><td></td><td>Use only in Class II safety cabinet</td></tr><tr><td>Phosphate buffer saline (PBS)</td><td>Fisher Scientific</td><td>12821680</td><td>pH 7.5, filtered before use</td></tr><tr><td>4% paraformaldehyde (PFA)</td><td>Sigma Aldrich</td><td>P6148</td><td>Once made can be stored at -20 &amp;deg;C in aliquotes for several months</td></tr><tr><td>4% formaldehyde&amp;nbsp; (FA)</td><td>Sigma Aldrich</td><td>F8775</td><td>Once made can be stored at -20 &amp;deg;C in aliquotes for several months</td></tr><tr><td>Ethanol, anhydrous denatured, histological grade</td><td>Sigma Aldrich</td><td>64-17-5</td><td>95, 75, 50% in PBS</td></tr><tr><td>0.01% Tween 20 in PBS</td><td>Sigma Aldrich</td><td>P9416</td><td>PBT</td></tr><tr><td>0.5% Triton X-100 in PBS</td><td>Sigma Aldrich</td><td>X100</td><td>PBX</td></tr><tr><td>2 N HCl</td><td>Sigma Aldrich</td><td>71826</td><td>caution extremely toxic</td></tr><tr><td>100 mM Tris-HCl&amp;nbsp;</td><td>Promega</td><td>H5121</td><td>pH adjusted to 8.5</td></tr><tr><td>hydrophobic barrier pen</td><td>Abcam</td><td>ab2601</td><td>for immunohistochemistry</td></tr><tr><td>Slide moisture chamber</td><td>Scientific Device Laboratory</td><td>197-BL</td><td></td></tr><tr><td>anti-5mC mouse antibody</td><td>Diagenode</td><td>C15200081</td><td>monoclonal primary antibody</td></tr><tr><td>Anti-5hmC antibody</td><td>Active Motif</td><td>39791</td><td>rabbit polyclonal primary antibody</td></tr><tr><td>Anti-5caC antibody</td><td>Active Motif</td><td>61225</td><td>rabbit polyclonal primary antibody</td></tr><tr><td>Peroxidase-conjugated anti-rabbit seconday antibody</td><td>Dako</td><td>K1497</td><td></td></tr><tr><td>555-congjuated goat anti-mouse antibody</td><td>Molecular probes</td><td>A-11005</td><td>&amp;nbsp;secondary antibody</td></tr><tr><td>10% BSA</td><td>Sigma Aldrich</td><td>A9418</td><td>Blocking solution</td></tr><tr><td>22 x 32 mm Glass cover slips</td><td>BDH</td><td>406/0188/24</td><td></td></tr><tr><td>Tyramide Signal Amplification System</td><td>Perkin Elmer</td><td>NEL741001KT</td><td>Other fluorochome conjugated seconday antibodies can be used for co-detection of oxi-5mC&amp;nbsp;</td></tr><tr><td>&amp;nbsp;Mounting Medium with DAPI</td><td>Vector Labs</td><td>H-1200</td><td></td></tr><tr><td>Colourless nail polish</td><td></td><td></td><td></td></tr><tr><td>chicken polyclonal anti-GFAP polyclonal antibody</td><td>Thermo Scientific</td><td>PA5-18598</td><td>1:400 dilution</td></tr><tr><td>anti-NeuN mouse monoclonal antibody</td><td>Merck milipore</td><td>MAB377B</td><td>1:400 dilution</td></tr></tbody></table>

detection of modified forms of cytosine using sensitive immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

뇌 조직 섹션에서 5hmC의 분포를 확인하기 위해, 우리는 특히이 마크와 상호 작용하지만 상업 항 5hmC 항체를 사용, 사후 유사 분열 뉴런, NeuN에 대한 마커이 후생 유전 학적 변형의 공동 검출을 수행되지 수정의 다른 형태와 시토신 (20), (25). 성인 뇌의 5hmC 및 5caC 분포의 면역 조직 화학적 분석은 눈에 띄는 5hmC 염색이 NeuN 양성 세포와 공동 지역화 반면, NeuN 음성 아교 세포가 유전자 5hmC (그림 1) (20)의 낮은 수준을 가지고 것으로 나타났습니다. 우리는 최근 구정에 의존 5mC 산화가 신경 줄기 세포 (NSCs의) 20의 혈통 사양 중 동작하는 것으로 나타났다. NSCs의에서 immunochemically 탐지에도 불구하고, 5fC 및 5caC는 신경 a를 향해 NSCs의 분화의 초기 단계에서 눈에 띄는 면역 염색을 전시<html> 차 아교 계통. 이 두 마크는 일시적으로 초기 신경 세포 및 교세포의 분화 (20)의 마커의 모양과 동시에 축적된다. 우리는 아교 분화 유도 3 일에서 NSCs을의 고정 문화에 아교 마커 GFAP에이 마크의 공동 염색을 수행 NSCs의 차별화에 5caC의 분포를 확인하십시오. NSCs의 성숙이나 성상 달리 (데이터 미도시), 우리는 이러한 배양에서 GFAP 발현 세포 (도 2) (20)의 비교적 큰 비율 5caC 강한 신호를 관찰 하였다. <img alt="그림 1" src="/files/ftp_upload/54416/54416fig1.jpg" /> 5hmC DAPI (파란색)으로 대조 성인 뇌 조직에서 NeuN (빨간색)와 (녹색). 개별 채널과 병합보기 그림 1. 공동 면역 염색이 표시됩니다. 스케일 바는 25 μm의 수 있습니다.&quot;빈&gt;이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오. <img alt="그림 2" src="/files/ftp_upload/54416/54416fig2.jpg" /> 그림 폐해 차별화의 3 일에서 NSC의 문화에서 GFAP와 5caC 2. 공동 면역 염색. 개별 채널과 병합 된 뷰가 표시됩니다. 스케일 바는 20 μm의 수 있습니다. <a href="https://www.jove.com/files/ftp_upload/54416/54416fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry at JoVE.com

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

민감한 면역를 사용하여 시토신의 수정 된 형태의 검출

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. ...

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7.2K 조회수.  Université Libre de Bruxelles. Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

비디오: Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

DNA 수정에 대 한 Immunostaining: 공초점 이미지의 계산 분석

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

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유전학

High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research. Mammalian DNA is enzymatically modified at the 5th carbon position of cytosine (C) residues to 5-mC, predominately in the context of CpG dinucleotides. 5-mC is amenable to enzymatic oxidation to 5-hmC by the Tet family of enzymes, which are believed to be involved in development and disease. Currently, the biological role of 5-hmC is not fully understood, but is generating a lot of interest due to its potential as a biomarker. This is due to several groundbreaking studies identifying 5-hydroxymethylcytosine in mouse embryonic stem (ES) and neuronal cells. Research techniques, including bisulfite sequencing methods, are unable to easily distinguish between 5-mC and 5-hmC . A few protocols exist that can measure global amounts of 5-hydroxymethylcytosine in the genome, including liquid chromatography coupled with mass spectrometry analysis or thin layer chromatography of single nucleosides digested from genomic DNA. Antibodies that target 5-hydroxymethylcytosine also exist, which can be used for dot blot analysis, immunofluorescence, or precipitation of hydroxymethylated DNA, but these antibodies do not have single base resolution.In addition, resolution depends on the size of the immunoprecipitated DNA and for microarray experiments, depends on probe design. Since it is unknown exactly where 5-hydroxymethylcytosine exists in the genome or its role in epigenetic regulation, new techniques are required that can identify locus specific hydroxymethylation. The EpiMark 5-hmC and 5-mC Analysis Kit provides a solution for distinguishing between these two modifications at specific loci. The EpiMark 5-hmC and 5-mC Analysis Kit is a simple and robust method for the identification and quantitation of 5-methylcytosine and 5-hydroxymethylcytosine within a specific DNA locus. This enzymatic approach utilizes the differential methylation sensitivity of the isoschizomers MspI and HpaII in a simple 3-step protocol. Genomic DNA of interest is treated with T4-BGT, adding a glucose moeity to 5-hydroxymethylcytosine. This reaction is sequence-independent, therefore all 5-hmC will be glucosylated; unmodified or 5-mC containing DNA will not be affected. This glucosylation is then followed by restriction endonuclease digestion. MspI and HpaII recognize the same sequence (CCGG) but are sensitive to different methylation states. HpaII cleaves only a completely unmodified site: any modification (5-mC, 5-hmC or 5-ghmC) at either cytosine blocks cleavage. MspI recognizes and cleaves 5-mC and 5-hmC, but not 5-ghmC. The third part of the protocol is interrogation of the locus by PCR. As little as 20 ng of input DNA can be used. Amplification of the experimental (glucosylated and digested) and control (mock glucosylated and digested) target DNA with primers flanking a CCGG site of interest (100-200 bp) is performed. If the CpG site contains 5-hydroxymethylcytosine, a band is detected after glucosylation and digestion, but not in the non-glucosylated control reaction. Real time PCR will give an approximation of how much hydroxymethylcytosine is in this particular site. In this experiment, we will analyze the 5-hydroxymethylcytosine amount in a mouse Babl/C brain sample by end point PCR.

The EpiMark 5-hmC and 5-mC Analysis Kit can be used to analyze and quantitate 5-methylcytosine and 5-hydroxymethylcytosine within a spe cific locus. The kit distinguishes 5-mC from 5-hmC by the addition of glucose to the hydroxyl group of 5-hmC via an enzymatic reaction utilizing &beta;-glucosyltransferase (T4-BGT). When 5-hmC occurs In the context of CCGG, this modification converts a cleavable MspI site to a non-cleavable site.

Balb / C 뇌 조직에 높은 민감도는 5 hydroxymethylcytosine 감지

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research.   Mammalian DNA is enzymatically ...

신경과학

Immunohistochemical Detection of 5-Methylcytosine and 5-Hydroxymethylcytosine in Developing and Postmitotic Mouse Retina

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a variety of human retinal degenerative diseases and pinpoint new treatment approaches. The nuclear architecture of mouse retina is organized in two different patterns: conventional and inverted. Conventional pattern is universal where heterochromatin is localized to the periphery of the nucleus, while active euchromatin resides in the nuclear interior. In contrast, inverted nuclear pattern is unique to the adult rod photoreceptor cell nuclei where heterochromatin localizes to the nuclear center, and euchromatin resides in the nuclear periphery. DNA methylation is predominantly observed in chromocenters. DNA methylation is a dynamic covalent modification on the cytosine residues (5-methylcytosine, 5mC) of CpG dinucleotides that are enriched in the promoter regions of many genes. Three DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) participate in methylation of DNA during development. Detecting 5mC with immunohistochemical techniques is very challenging, contributing to variability in results, as all DNA bases including 5mC modified bases are hidden within the double-stranded DNA helix. However, detailed delineation of 5mC distribution during development is very informative. Here, we describe a reproducible technique for robust immunohistochemical detection of 5mC and another epigenetic DNA marker 5-hydroxymethylcytosine (5hmC), which colocalizes with the &#34;open&#34;, transcriptionally active chromatin in developing and postmitotic mouse retina.

The objective of this report is to describe the protocol for robust immunohistochemical detection of epigenetic markers, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in developing and postmitotic mouse retina.

5-Methylcytosine 개발에 5-Hydroxymethylcytosine의 Immunohistochemical 검출 및 Postmitotic 마우스 망막

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a ...

발생학

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

형광에 의한 신경 조직의 영구 DNA 바이러스의 게놈 및 성적 증명서의 검출 현장에서 하이브리드 면역 염색과 결합

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

Ubiquitylated, SUMOylated 및 ADP-Ribosylated DNA-Protein Crosslinks 측정

Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, methyltransferases, etc.) malfunctioning, or exogenous agents such as chemotherapeutics and crosslinking agents. Once DPCs are induced, several types of post-translational modifications (PTMs) are promptly conjugated to them as early response mechanisms. It has been shown that DPCs can be modified by ubiquitin, small ubiquitin-like modifier (SUMO), and poly-ADP-ribose, which prime the substrates to signal their respective designated repair enzymes and, in some cases, coordinate the repair in sequential manners. As PTMs transpire quickly and are highly reversible, it has been challenging to isolate and detect PTM-conjugated DPCs that usually remain at low levels. Presented here is an immunoassay to purify and quantitatively detect ubiquitylated, SUMOylated, and ADP-ribosylated DPCs (drug-induced topoisomerase DPCs and aldehyde-induced non-specific DPCs) in vivo. This assay is derived from the RADAR (rapid approach to DNA adduct recovery) assay that is used for the isolation of genomic DNA containing DPCs by ethanol precipitation. Following normalization and nuclease digestion, PTMs of DPCs, including ubiquitylation, SUMOylation, and ADP-ribosylation, are detected by immunoblotting using their corresponding antibodies. This robust assay can be utilized to identify and characterize novel molecular mechanisms that repair enzymatic and non-enzymatic DPCs&#160;and has the potential to discover small molecule inhibitors targeting specific factors that regulate PTMs to repair DPCs.

저자 스포트라이트: DNA 단백질 교차결합의 정량적 검출 및 번역 후 변형(Post-Translational Modifications)  

The present protocol highlights a modified method to detect and quantify DNA-protein crosslinks (DPCs) and their post-translational modifications (PTMs), including ubiquitylation, SUMOylation, and ADP-ribosylation induced by topoisomerase inhibitors and by formaldehyde, thereby allowing the study of the formation and repair of DPCs and their PTMs.

DNA-단백질 가교 및 번역 후 변형의 정량적 검출

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, ...

생화학

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

게놈 DNA에서 도보로 5 hydroxymethylcytosine의 선택적 캡처

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

생물학

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

신경 줄기 세포와 쥐의 뇌에서 5-하이드록시메틸시토신의 검출

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&#38;E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&#38;E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

qPCR에 포르말린 고정, 파라핀 - 임베디드 생검 조직에서 Cytomegaloviral DNA의 검출에 대한 민감하고 빠른 방법입니다

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...

민감한 면역를 사용하여 시토신의 수정 된 형태의 검출

요약

더 많은 비디오 탐색

이 비디오의 챕터

DNA 수정에 대 한 Immunostaining: 공초점 이미지의 계산 분석

Balb / C 뇌 조직에 높은 민감도는 5 hydroxymethylcytosine 감지

5-Methylcytosine 개발에 5-Hydroxymethylcytosine의 Immunohistochemical 검출 및 Postmitotic 마우스 망막

형광에 의한 신경 조직의 영구 DNA 바이러스의 게놈 및 성적 증명서의 검출 현장에서 하이브리드 면역 염색과 결합

DNA-단백질 가교 및 번역 후 변형의 정량적 검출

DNA-단백질 가교 및 번역 후 변형의 정량적 검출

DNA-단백질 가교 및 번역 후 변형의 정량적 검출

게놈 DNA에서 도보로 5 hydroxymethylcytosine의 선택적 캡처

신경 줄기 세포와 쥐의 뇌에서 5-하이드록시메틸시토신의 검출

qPCR에 포르말린 고정, 파라핀 - 임베디드 생검 조직에서 Cytomegaloviral DNA의 검출에 대한 민감하고 빠른 방법입니다