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 border="0" cellpadding="0" cellspacing="0" width="1281">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:388px;">Coplin jar</td>
			<td style="width:180px;">Cole-Parmer</td>
			<td style="width:119px;">UY-48585-30</td>
			<td style="width:595px;">Glass made</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xylene</td>
			<td></td>
			<td></td>
			<td>Use only in Class II safety cabinet</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate buffer saline (PBS)</td>
			<td>Fisher Scientific</td>
			<td>12821680</td>
			<td>pPH 7.5, filtered before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4% paraformaldehyde (PFA)</td>
			<td>Sigma Aldrich</td>
			<td>P6148</td>
			<td>Once made can be stored at -20&#176;C in aliquotes for several months</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4% formaldehyde&#160; (FA)</td>
			<td>Sigma Aldrich</td>
			<td>F8775</td>
			<td>Once made can be stored at -20&#176;C in aliquotes for several months</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol, anhydrous denatured, histological grade</td>
			<td>Sigma Aldrich</td>
			<td>&#160;64-17-5&#160;&#160;</td>
			<td>95, 75, 50 % in PBS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.01 % Tween 20 in PBS</td>
			<td>Sigma Aldrich</td>
			<td>P9416</td>
			<td>PBT</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5% Triton X-100 in PBS</td>
			<td>Sigma Aldrich</td>
			<td>X100</td>
			<td>PBX</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2 N HCl</td>
			<td>Sigma Aldrich</td>
			<td>71826</td>
			<td>caution extremely toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100 mM Tris-HCl&#160;</td>
			<td>Promega</td>
			<td>H5121</td>
			<td>PH adjusted to 8.5</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hydrophobic barrier pen</td>
			<td>Abcam</td>
			<td>ab2601</td>
			<td>for immunohistochemistry</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Slide moisture chamber</td>
			<td>Scientific Device Laboratory</td>
			<td>197-BL</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-5mC mouse antibody</td>
			<td>Diagenode</td>
			<td>C15200081</td>
			<td>monoclonal primary antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-5hmC antibody</td>
			<td>Active Motif</td>
			<td>39791</td>
			<td>rabbit polyclonal primary antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-5caC antibody</td>
			<td>Active Motif</td>
			<td>61225</td>
			<td>rabbit polyclonal primary antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Peroxidase-conjugated anti-rabbit seconday antibody</td>
			<td>Dako</td>
			<td>K1497</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">555-congjuated goat anti-mouse antibody</td>
			<td>Molecular probes</td>
			<td>A-11005</td>
			<td>&#160;secondary antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10% BSA</td>
			<td>Sigma Aldrich</td>
			<td>A9418</td>
			<td>Blocking solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">22x32mm Glass cover slips</td>
			<td>BDH</td>
			<td>406/0188/24</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;Mounting Medium with DAPI</td>
			<td>Vector Labs</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Colourless nail polish</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">chicken polyclonal anti-GFAP polyclonal antibody</td>
			<td>Thermo Scientific</td>
			<td style="width:119px;">PA5-18598</td>
			<td>1:400 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-NeuN mouse monoclonal antibody</td>
			<td>Merck milipore</td>
			<td style="width:119px;">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://jove.unicartagenaproxy.elogim.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. ...

detection-modified-forms-cytosine-using-sensitive

Research

JoVE Journal

Biology

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

Thin Sectioning of Slice Preparations for Immunohistochemistry

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been preserved, freshly removed, or excised but kept viable, as in slice preparations of brain tissue.  Subsequent microscopic studies of this material can be challenging, as the tissue samples may be difficult to handle.  Demonstrated here is a method for obtaining thin cryostat sections of tissue with a thickness that may range from 0.2-5.0 mm.  We routinely cut 400 micron thick Vibratome brain slices serially into 5-10 micron coronal cryostat sections.  The slices are typically first used for electrophysiology experiments and then require microscopic analysis of the cytoarchitecture of the region from which the recordings were observed.  We have constructed a simple device that allows controlled and reproducible preparation and positioning of the tissue slice.  This device consists of a cylinder 5 cm in length with a diameter of 1.2 cm, which serves as a freezing stage for the slice.  A ring snugly slides over the cylinder providing walls around the slice allowing the tissue to be immersed in freezing compound (e.g., OCT).  This is then quickly frozen with crushed dry ice and the resulting  wafer  can be position easily for cryostat sectioning.  Thin sections can be thaw-mounted onto coated slides to allow further studies to be performed, such as various staining methods, in situ hybridization, or immunohistochemistry, as demonstrated here.

The present method allows reproducible cryostat sectioning of small, difficult-to-manage, tissue pieces, such as biopsies and brain slices. We utilize a simple aluminum freezing stage to facilitate handling of tissue and a standard cryostat to routinely produce 5-10 micron serial sections from 400 micron thick brain slices.

Immunohistochemistry에 대한 슬라이스 준비 씬 Sectioning

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been ...

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생물학

Preventing the Spread of Malaria and Dengue Fever Using Genetically Modified Mosquitoes

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission.   Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced, as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission. Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

유전자 변형 모기를 사용하여 말라리아와 뎅그열 피버의 확산을 방지

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission.   Population ...

Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific antibodies. There are two methods: the direct and indirect method. In this experiment, we will only describe the use of indirect IHC staining. Indirect IHC staining utilizes highly specific primary and biotin-conjugated secondary antibodies. Primary antibodies are utilized to discretely identify proteins of interest by binding to a specific epitope, while secondary antibodies subtract for non-specific background staining and amplify signal by forming complexes to the primary antibody. Slides can either be generated from frozen sections, or paraffin embedded sections mounted on glass slides. In this protocol, we discuss the preparation of paraffin-embedded sections by dewaxing, hydration using an alcohol gradient, heat induced antigen retrieval, and blocking of endogenous peroxidase activity and non-specific binding sites. Some sections are then stained with antibodies specific for T cell marker CD8 and while others are stained for tyrosine hydroxylase. The slides are subsequently treated with appropriate secondary antibodies conjugated to biotin, then developed utilizing avidin-conjugated horseradish peroxidase (HRP) with Diaminiobenzidine (DAB) as substrate. Following development, the slides are counterstained for contrast, and mounted under coverslips with permount. After adequate drying, these slides are then ready for imaging.

Immunohistochemistry : 벡터 연구소에서 Vectastain ABC 키트를 사용하여 파라핀 섹션

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific ...

Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, suprabasal, and stratum corneum layers. The precise localization within the epidermis of a particular protein lends clues toward its functional role within the epidermis. In this chapter, we describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory. Paraffin sections generally provide much better morphology, hence, superior results and photographs; however, not all antibodies will work with the harsh fixation and treatment involved in their processing. Therefore, the protocol for frozen sectioning is also included. Within paraffin sectioning, two fixation protocols are described (Bouin's and paraformaldehyde); the choice of fixative will be directly related to the antibody specifications and may require another fixing method.

We describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory.

형광 항체를 사용하여 마우스 표피의 파라핀 섹션에 Immunohistochemistry

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, ...

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

Drosophila 애벌레 NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Optical Recording of Electrical Activity in Guinea-pig Enteric Networks using Voltage-sensitive Dyes

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons (10 to 25 &mu;m in diameter) are arranged in plexuses that are confined to distinct planes of the gut wall 1; the myenteric plexus can be found between the longitudinal and circular muscle layers, and the submucous plexus between the circular muscle layer and the mucosa. Since the effector systems for these plexuses (transporting epithelium, endocrine cells, immune elements, blood vessels and smooth muscle) are also contained within the gut wall, semi-intact preparations can be dissected that preserve individual components of different reflex pathways. The behavior of the effector systems is controlled by the submucous and myenteric plexuses acting in concert. Therefore, detailed knowledge of synaptic interactions within and between ganglia, and of communication between the plexuses, is essential for understanding normal gastrointestinal function. The ENS, as an intact nervous system, is a unique experimental model in which one can correlate molecular and cellular events with the electrical behavior of the neuronal network and its physiological outputs. Because of the quasi-two-dimensional organization of its plexuses, the ENS is particularly well suited for the study of neural networks using multiple site optical recording techniques that employ voltage-sensitive dyes 2,7,8,9. We will illustrate here the use of a relatively new naphthylstyryl-pyridinium dye (di-4-ANEPPDHQ) 3 that offers multiple advantages over its predecessors, including very low phototoxicity, slow rate of internalization, and remarkable chemical stability. When used in conjunction with a camera that permits sub-millisecond time resolution, this dye allows us to monitor the electrical activity of all the neurons in the field of view with a maximal spatial resolution of ~ 2.5 &mu;m at 100X magnification. At lower magnification (10X or 20X), the sacrifice of single-cell resolution is compensated by a gain in perspective, revealing the intricacies of the inter-ganglionic circuitry.

This protocol illustrates how voltage-sensitive dyes enable optical recording of electrical activity from intact neural networks such as the plexuses of the guinea-pig enteric nervous system, with an adjustable resolution that ranges from single-cells to multi-ganglionic circuitry.

전압에 민감한 염료를 사용하여 기니 돼지 장용 네트워크에서 전기 활동의 광학 녹음

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons ...

Drosophila Pupal Abdomen Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic morphologies 1-3. During pupation, which spans approximately 96 hours (at 25 &deg;C), proliferating populations of imaginal cells replace the larval epidermis to generate the adult abdominal segments. These imaginal cells, born during embryogenesis, exist as lateral pairs of histoblast nests in each abdominal segment of the larvae. Four pairs of histoblast nests give rise to the adult dorsal cuticle (anterior and posterior dorsal nests), the ventral cuticle (ventral nests) and the spiracles associated with each segment (spiracle nests) 4. Upon puparation, these diploid cells (distinguishable by size from the larger polyploid larval epidermal cells- LECs) begin a stereotypical process of proliferation, migration and replacement of the LECs. Various molecular and genetic tools can be employed to investigate the contributions of genetic pathways involved in morphogenesis of the adult abdomen. Ultimate adult phenotypes are typically analyzed following dissection of adult abdominal cuticles. However, investigation of the underlying molecular processes requires immunohistochemical analyses of the pupal epithelium, which present unique challenges. Temporally dynamic morphogenesis and the interactions of two distinct epithelial populations (larval and imaginal) generate a fragile tissue prone to excessive cell loss during dissection and subsequent processing. We have developed methods of dissection, fixation, mounting and imaging of the Drosophila pupal abdominem epithelium for immunohistochemical studies that generate consistent high quality samples suitable for confocal or standard fluorescent microscopy.

Drosophila Pupal Abdomen Immunohistochemistry

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

Drosophila Pupal의 복부 Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic ...

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 ...

Colorimetric Detection of Bacteria Using Litmus Test

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.

We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads.

리트머스 테스트를 사용하여 박테리아의 비색 검출

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. ...

Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between defective mitophagy and various human diseases, including neurodegenerative diseases, cancer, and metabolic diseases. In addition, recent studies have shown that mitophagy is involved in normal cellular processes, such as differentiation and development. However, the precise role of and molecular mechanisms underlying mitophagy require further study. Therefore, it is critical to develop a robust and convenient method for measuring changes in mitophagy activity. Here, we describe a detailed protocol for quantitatively assessing mitophagy activity through flow cytometry using the mitochondria-targeted fluorescent protein Keima (mt-Keima). This flow cytometry assay can analyze mitophagy activity more rapidly and sensitively than conventional microscopy- or immunoblotting-based methods. This protocol can be applied to analyze mitophagy activity in various cell types.

Mitophagy, the selective degradation of mitochondria, has been implicated in mitochondrial homeostasis and is deregulated in various human diseases. However, convenient experimental methods for measuring mitophagy activity are very limited. Here, we provide a sensitive assay for measuring mitophagy activity using flow cytometry.