우리는 자신의 기술 지원을 트렌트 워 감사하고 싶습니다. 우리는 또한 아픈 아이들이 프로젝트에 대한 자신의 관대 한 자금에 대한 치료 선천성 근육 영양 장애 (CMD)의 병원에서 소아과를 인정합니다.

한천 사출 플레이트 1. 준비 (시간 : 45 분) <ol><li> E3 미디어의 2 % ~ 3 % 아가로 오스를 삶아 및 솔루션은 벤치에 약간 냉각 할 수 있습니다. 참고 제조되는 분사 플레이트의 수는 필요 아가 로스의 양을 지시한다. 각 분사 플레이트 아가 용액 약 35 ㎖에 필요하다. </li><li> 끓는 후, 원하는 온도가 사출 금형 제조 업체의 지침에 따라 (예., 45 ℃로)에 도달 할 때까지 아가로 오스를 냉각 할 수 있습니다. </li><li> 100mm 접시에 아가로 오스 냉각의 약 35 ml에 붓고. </li><li> 솔루션으로 선호하는 사출 금형의 한쪽 끝을 놓고, 다음 (이 기포의 발생을 줄이는 데 도움이됩니다) 아가로 오스 솔루션에 금형의 나머지를하다. </li><li> 아가로 오스 용액을 약 30 분 동안 하나 실온에서 4 ° C를 공고히 할 수 있습니다. </li><li> 고체 아가에서 금형의 일단을 분리 주걱을 사용한다. 천천히 m의 나머지를 제거<html> 늙은. </li></ol> 에반스 블루 염료 (EBD) 사출 믹스 2. 준비 (시간 : 30 분) <ol><li> 1X 링거액에 EBD의 1 %의 주식을 확인 (5 mM의 KCl을 155 mM의 NaCl을 2 mM의 염화칼슘 2 1 밀리미터의 MgCl 2, 2 밀리미터 나 2 HPO 4, 10 mM의 HEPES 10 mM의 포도당, 7.2의 pH), 이는 실온에서 저장 될 수있다. </li><li> 25 밀리그램에서 MW 10,000 kDa의 -dextran 형광 염료의 주식 솔루션 (FITC)를 확인 / ㎖ -20 ℃에서 1X 링거액 및 저장소 </li><li> (. 즉, 100 μL의 최종 작업 볼륨 : FITC 덱스 트란 주식의 90 μL에 1 % EBD 10 μl를 혼합) FITC-덱스 트란 주식의 원액을 직접 0.1 %에 EBD를 희석하여 주입 믹스를 준비합니다. </li><li> 철저하게 소용돌이 주입 믹스 (이 녹색으로 변합니다) 및 알루미늄 호일에 주입 혼합 튜브를 배치하여 직접 빛이 닿지 않는 곳에 보관. </li></ol> 3. EBD 주입 준비 (시간 : 약 30 분) ENT &quot;&gt; 참고 : 프로토콜 3-7 일 포스트 수정 (DPF)에서 유충으로 가장 적합합니다. <ol><li> RT에 대한 사전 따뜻한 주입 판. </li><li> 금속 접시에 미세 조작기를 배치하여 사출 장치를 설정하고 현미경 옆에 사출에 사용되는 서있다. 공기 구동 미세 주입 컨트롤러를 켭니다. 참고 : 선호 분사 시스템은 실험실에 따라 다릅니다 및 분석의 결과를 변경해서는 안됩니다. </li><li> 위로 EBD 믹스의 약 2-4 μL와 주사 바늘을 입력합니다. </li><li> EBD 믹스의 약 5 NL에 주입 볼륨을 보정합니다. 주 : 주입량 교정 교정 방법에 의존 할 것이다. 가스 압력이 인젝터 마이크로 미터를 사용하여 볼륨을 통해 볼 루스 보정 주입 부피를 필요로하는 반면, 피스톤 구동 주입 직접 주어진 주입 부피로 설정 될 수있다. </li><li> 1X 링거액 젖은 주입 판과는 우물에서 초과 제거합니다. </li><li> 0.04 %의 에틸 3- 아미노 벤조 에이트의 메탄 샐 사전 치료 유충분사의 시작에 앞서 유충 고정화 1X 링거액 희석 t (tricaine). 참고 : 적절한 주입 잔여 움직임 어렵 기 때문에 유충을 보장하는 것은 완전히 immotile 있습니다 중요하다. </li><li> 유리 피펫을 사용하여 한천 주입 판 우물에 마취 애벌레를 놓습니다. 유충이 완전히 잘 내에서 자신의 측면에 누워 있는지 확인합니다. 참고 : 물론 당 유충의 수는 실험에 달려있다. </li><li> 유충이 웰에 넣어 후 웰 내에 유충 움직임을 최소화하기 위해 과량의 링거액을 제거한다. 그 애벌레가 탈수되지 않도록 용액의 잔류량을 남겨주세요. </li></ol> EBD와 Zebrafish의 유충 4. 심막 주입 (시간 : 유충 주입의 수에 따라, 약 1-3 시간) <ol><li> 주사가 수행 될 해부 범위에 유충을 포함하는 주입 판을 놓습니다. </li><li> 포함하는 주입 피펫 바늘의 위치를EBD는 제브라 피쉬 애벌레를 통해 섞는다. </li><li> 다시 위치를 회전시킴으로써 분사 플레이트 정도로 주사 바늘은 유충의 중심부 근처이며, 대략 45 ° 복부 전후방 축으로부터. </li><li> 정맥 처음 지느러미 방향으로 돌고 노른자 (그림 1)의 전방 부분에서 정맥의 영역에서 일반 추기경 정맥 (CCV)에 주사 바늘을 ​​삽입합니다. 참고 : 최대 40 배까지의 배율이 명확하게 CCV를 참조하는 것이 유용 할 수 있습니다. </li><li> EBD 믹스 5 NL을 주입하고 EBD 믹스 즉시 누출을 최소화하기 위해 5-8 초 동안 위치에 주사 바늘을 유지. 참고 : 좋은 주입은 심장 챔버에서 볼 수 염료 착색 (그림 1)을해야합니다. EBD 믹스는 심장에서 관찰되지 않으면, EBD 믹스 추가 5 NL 염료 흡수를 유도하기에 충분할 수있다 주입. 또한, 배아는 폐기 될 수있다. 참고 : 어떤 경우에는 심장이 박동을 중지 할 수 있습니다. 이 O를하는 경우ccurs, 20 ~ 40 초 동안 유충을 모니터링하는 것을 계속한다. 염료가 순환 시스템을 통해 이동함에 따라 일반적으로 심장 박동이 다시 시작됩니다. </li><li> 다음 유충 반복에 이동합니다. </li><li> 바로 주입 (그림 2) 후 혈관에 FITC-덱스 트란의 존재를 관찰하여 성공적으로 주입 된 배아를 식별합니다. </li></ol> 5. 배양 및 EBD 통풍 관 (시간 : 4 ~ 6 시​​간) <ol><li> 유충의 원하는 번호가 주입 된 후, 반환 100mm 요리에 tricaine없이 1X 링거액에 유충을 주입. </li><li> 알루미늄 호일에 싸서 요리를 유지합니다. 주 : 어두운 주입 유충 크게 늘리지 생존율을 증가시키고, 신호 강도에 큰 일관성을 보장한다. 알루미늄 호일로 포장 유충 인큐베이터 외부의 시간 동안 특히 중요하다. </li><li> 충분한 EBD 흡수를 보장하기 위해 유충은 4 ~ 6 시간 동안 28.5 ℃에서 부화 할 수 있습니다. </li></OL&gt; 근육에 EBD 6. 시각화 (시간 : 유충 주입과 현미경의 종류의 수에 따라, 예상 0.5-3 시간) <ol><li> 이전 영상에 움직임을 방지하기 위해 0.04 %의 유충과 tricaine 마취. </li><li> EBD 흡수가 골격 근육 (그림 3)에서 발생하는 경우 적색 형광에서보기 유충 확인합니다. </li></ol>

<ol>
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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hooked!+Modeling+human+disease+in+zebrafish.">Hooked! Modeling human disease in zebrafish.</a> J Clin Invest. 122, 2337-2343 (2012).</li><li>Majczenko, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dominant+Mutation+of+CCDC78+in+a+Unique+Congenital+Myopathy+with+Prominent+Internal+Nuclei+and+Atypical+Cores.">Dominant Mutation of CCDC78 in a Unique Congenital Myopathy with Prominent Internal Nuclei and Atypical Cores.</a> Am J Hum. , (2012).</li><li>Wooddell, C. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Evans+blue+dye+to+compare+limb+muscles+in+exercised+young+and+old+mdx+mice.">Use of Evans blue dye to compare limb muscles in exercised young and old mdx mice.</a> Muscle Nerve. 41, 487-499 (2010).</li><li>Hall, T. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+candyfloss+mutant+implicates+extracellular+matrix+adhesion+failure+in+laminin+alpha2-deficient+congenital+muscular+dystrophy.">The zebrafish candyfloss mutant implicates extracellular matrix adhesion failure in laminin alpha2-deficient congenital muscular dystrophy.</a> Proc Natl Acad Sci U S A. 104, 7092-7097 (2007).</li></ol>

저자는 더 경쟁 재정적 이익 또는 관심의 다른 충돌이 없습니다.

제브라 피쉬는 신경 근육 질환 2,29의 연구를위한 강력한 도구로 부상하고있다. 현재까지, 제브라 피쉬 시스템은 새로운 근육 질환을 일으키는 돌연변이를 확인 16,17,30 신규 pathomechanisms 18을 해명하고, 잠재적으로 새로운 치료약 (12, 24)를 식별하는 데 사용되어왔다. 이러한 집단적인 노력은 인간의 신경 근육 질환을 모델링 할 수있는 제브라 피쉬의 유틸리티를 설립했다. 그러나, 제브라 피쉬와 포유 동물 모델로 만든 진보에도 불구하고, 신경 근육 조건의 넓은 스펙트럼 내에서 환자에 대한 제한적인 치료 옵션이 있습니다. 따라서, 수요는 파괴적인 질병이 그룹에 대한 치료 개발을 위해 존재한다. 치료법이 요구 평행하면 새로운 동물 모델 및 추정 치료 전략을 검증하는 실험 진행 혁신뿐만 아니라 엄격한 분석을위한 대응이 필요하다. EBD 분석은 일반적으로 마우스 모델에서 사용연구 조직과 뇌, 심장에서 세포 손상 및 골격 근육 27,31. 특히, EBD 근육 세포막 불안정성의 정도를 표시하고 8 손상 근이영양증 다양한 아형의 마우스 모델에서 광범위하게 사용된다. 근육 세포막 손상을 드러내는 EBD의 사용은 인간의 질병 상태 (9) 동물 모델의 유사성을 확립지지 파라미터이다. 마우스에 EBD의 힘은 개발 및 신경 근육 질환의 제브라 피쉬 모델에 EBD를 적용, 우리 자신을 포함한 여러 실험실을 주도하고있다. EBD 인해 분석의 적용으로,이 기술은 활발 인간 질병 상태에 11,15,22,24,32 제브라 피쉬 모델을 확증하기 위해 구현되고있다. 손상된 근육 막과 유충은 근육 섬유 내의 EBD 흡수하기 때문에 적색 형광을해야합니다. 개별 근섬유 내의 섬유 간 공간에 형광 관찰 아니지만 역시 t의 기저막로부터 분리 섬유의 정보 일 수있다유용한 진단 세부 사항을 제공하는 멤브레인 손상의 그는 부재. EBD 분석은 동물 모델 검증을 넘어 잠재적 인 응용 프로그램이 있습니다. 우리의 실험실에서 노력은 최근 EBD 분석은 잠재적으로 새로운 치료 약물 (24)의 유효성을 검사에 도움이되도록 증명하고있다. 잠재적 인 치료 치료를 감소 시키거나 관련 치료 활동 (8)을 의미 할 수 있습니다 신경 근육 질환 모델에 EBD 흡수를 폐지 여부 확인. 이러한 유형의 분석 치료제 메커니즘 (들)을 설정하는 데 도움 EBD 및 분석 애플리케이션을 확대 할 수있다. 많은 기술과 마찬가지로, EBD 분석은 실험 설계 및 연습 동안 관찰 할 몇 가지주의 사항을 가지고있다. 예를 들어, 나이 인해 조직의 비후에 CCV를 식별하기 어려울 수있다. 또한, 실험 횟수를 줄이고 다수의 유충 수험 할 필요성을 증가 전과 심낭 주입 중에 제조에서 유충을 손상하기 쉽다.손상된 근육 EBD을 차지할 수 게다가, 처리 및 사출 동안 유충 짓을 물리적 손상은 오탐 (false positive)이 발생할 수 있습니다. 이러한 장애들을 극복하기 위해, 우리는 사출 전에 후속 분석 직후 성공적인 염료 주입과 유충 쉽고 신뢰할 수 있도록 식별이 비디오 문서 공동 사출 전략을 설명 하였다. 전에 근육 섬유로의 흡수로 혈관에 EBD의 확인을 허용하여 성공적으로 주입을위한 공동 분사 제어 FITC-덱스 트란. EBD 형광은 근육 섬유에서 수집되지 않을 경우 몇 시간 후 애벌레 매우 확산되면서 특히 유용 할 수있다; 이와 같이, 탐지하기 어려울 수있다. 또한, CCV 누락 및 노른자 또는 신체 공동으로 EBD를 주입하면, 배양 한 후, 손상된 근육 섬유에 의해 흡수의 감소 가능성과 아직 배아를 제어 할 유사한 확산 붉은 형광이 발생할 수 있습니다. 종합적으로,이 동굴ATS는 EBD 주입이 일관되고 신뢰할 수있는 결과를 달성하기 위해 인내와 연습이 필요 좋습니다. 모든에서는 제브라 애벌레 EBD 분석을 수행 실용적이고 간단한 방법을 설명한다. 지금까지 모델 시스템으로서 지브라 피쉬의 사용은 특히 인간의 질환 모델로서, 급속하게 확장되고있다. 이 확장은 제브라 피쉬 시스템의 현재 장점을 개선 실험 기술의 지속적인 개발 및 수정에 부분적으로 기인한다. EBD 주입 기술은 검증 및 제브라 피쉬 근육 질환 모델 연구를위한 연구자의 무기에 추가하고 강력한 도구를 제공합니다. 이 기술의 지속적인 구현 및 수정은 새로운 치료 전략뿐만 아니라 질병을 일으키는 메커니즘을 밝히기 도울 수있는 잠재력을 가지고있다.

Muscular dystrophies constitute a group of prevalent and heterogeneous human muscle diseases with specific histopathological features1,2. Symptoms typically associated with this devastating group of diseases include muscle weakness, muscle degeneration, loss of motility, and early mortality1,3. The primary pathomechanisms of muscular dystrophies are the loss of proteins that stabilize the sarcolemma, anchor transmembrane complexes, and mediate cell signaling across the membrane4-6. For example, complete loss of the protein dystrophin, a primary scaffold protein of the dystrophin-glycoprotein complex, results in destabilization of the muscle membrane in Duchenne muscular dystrophy7. Due to the fact that most muscular dystrophies result from mutations in proteins that participate in the link between the extracellular matrix and the sarcolemmal cytoskeleton, a common observation at the cellular level is the loss of sacrolemmal integrity8,9. This understanding of the primary pathomechanism(s) associated with muscular dystrophies is the product of numerous years of research employing animal model systems2,10-15. However, despite advances in the field, there are still limited therapeutic options for treatment or management of the range of dystrophy subtypes. This limitation of viable therapies is due to several key factors: 1) the difficulty of gene therapy strategies, 2) a high frequency of de-novo disease cases and the corresponding lack of translatable animal models, and 3) the lack of rigorous strategies to test the physiological consequences of putative therapeutic agents with clear and reproducible outcome measures.
To overcome some of these limitations, numerous labs including our own are employing zebrafish as a system to model and study human neuromuscular diseases2. To date, zebrafish have proven a valuable tool in disease research. The zebrafish model has been used to identify and validate novel human disease causing mutations16,17, elucidate uncharacterized disease causing mechanisms17,18, and identify novel therapeutic strategies12,19. These advances were made, in part, by the canonical strengths of the zebrafish system such as their optical clarity, ease of genetic manipulation, and ability to breed in large numbers20. Zebrafish have additionally proven amendable to large-scale drug screens21, a valuable method for the identification of novel therapeutics22-24. Regarding muscle disease research, these strengths are complemented by the ability to isolate single zebrafish skeletal muscle fibers via dissociation25 and by the ability to examine myofiber organization in vivo using the optical phenomenon called birefringence26, which collectively allows for rapid determination of macroscopic muscle integrity. Regardless of these available utilities, further tool development is continuously required to advance investigation.
We, and others, have adapted a protocol for EBD injection and analysis in the zebrafish model. EBD is a vital, cell permeable dye that is taken up by damaged, degenerating, or apoptotic cells and then visualized under fluorescence27. To date, EBD analysis has extensively been used to analyze muscle membrane integrity in mouse models of skeletal muscle and heart diseases8,9,27. However, in mammalian preparations, harvested muscle typically requires laborious sectioning or dissection prior to analysis. In zebrafish, direct analysis is possible in high numbers using live and intact animals. In this video article, we will demonstrate the methodology to perform EBD injection and analysis in live zebrafish larvae, with representative images of EBD uptake in the zebrafish dystrophy mutant line sapje15,28. Furthermore, we present a co-injection strategy that allows for increased quantification of EBD preparations.

<table border="0" cellpadding="0" cellspacing="0" width="801">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Fluorescein isothiocyanate-dextran MW 10,000</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">FD10S</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Evan&#39;s Blue Dye</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">E2129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Ethyl 3-aminobenzoate methanesulfonate salt</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">A5040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">100 mm Petri dish</td>
			<td style="width:173px;">Fischerbrand</td>
			<td style="width:140px;">FB0875712</td>
			<td>Injection mold base</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:321px;">Thin wall glass capillaries</td>
			<td style="width:173px;">World Precision Instruments</td>
			<td style="width:140px;">TW100F-4</td>
			<td>For Injection needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Agarose</td>
			<td style="width:173px;">Bioshop</td>
			<td style="width:140px;">AGA001</td>
			<td>Injection mold</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Microinjection mold</td>
			<td style="width:173px;">Adaptive Science Tools</td>
			<td style="width:140px;">TU-1</td>
			<td>Injection mold</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Sodium chloride</td>
			<td style="width:173px;">Bioshop</td>
			<td style="width:140px;">SOD001</td>
			<td>Ringer&#39;s solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Potassium chloride</td>
			<td style="width:173px;">Bioshop</td>
			<td style="width:140px;">POC888</td>
			<td>Ringer&#39;s solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Magnessium chloride hexahydrate</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">M2670</td>
			<td>Ringer&#39;s solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Sodium phosphate monobasic monohydrate</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">S9638</td>
			<td>Ringer&#39;s solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">HEPES</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">H4034</td>
			<td>Ringer&#39;s solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Glucose</td>
			<td style="width:173px;">BioBasic</td>
			<td style="width:140px;">GB0219</td>
			<td>Ringer&#39;s solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Calcium chloride</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">C1061</td>
			<td>Ringer&#39;s solution</td>
		</tr>
	</tbody>
</table>

analysis of zebrafish larvae skeletal muscle integrity with evans blue dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

EBD 주입 혼합 3 DPF에서 sapje 동형 접합 돌연변이와 야생 형 형제의 CCV에 주입했다. 심장 챔버 (그림 1B)를 가득 주입 후 FITC - 덱스 트란 녹색 형광 아래에있는 혈관에 (그림 2) 시각화하여 성공적으로 주입을 분석 하였다. 4 시간의 잠복기 후, EBD 흡수는 형광 현미경을 사용하여 체절 수준에서 조사 하였다. sapje 동형 접합 돌연변이가 근육 막 (15) (그림 3B)에 손상을 나타내는, EBD 흡수를 보여 주었다 반면 야생 형 형제, 눈에 보이는 근육 섬유 (그림 3A) 내에 EBD 형광을 나타내지 않았다. <img alt="그림 1" src="/files/ftp_upload/53183/53183fig1.jpg" /> 제브라 피쉬 엠브리의 일반 추기경 정맥 (CCV)에 그림 1. 주입 EBD 주입 믹스오. (A) Uninjected 배아. 화살표는 CCV 주입을위한 이상적인 위치를 나타낸다. (B) CCV에 성공 주입. 염료는 심장 챔버 (화살표)를 입력하고 혈관을 통해 펌핑하기 시작합니다. (C) 실패 CCV 주입 일부 또는 배아 (화살표)의 난황을 입력 염료의 모든 발생합니다. <a href="https://www.jove.com/files/ftp_upload/53183/53183fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" src="/files/ftp_upload/53183/53183fig2.jpg" /> 그림 2. 배아는 즉시 주입 전에 EBD 흡수에 다음과 같은 혈관을 통해 녹색 형광 아래 FITC-덱스 트란 분포를 관찰하여 성공적으로 주입 정렬 할 수 있습니다.이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오. <img alt="그림 3" src="/files/ftp_upload/53183/53183fig3.jpg" /> 그림 3 : EBD는 손상된 세포막과 섬유에 의해 흡수 될 것입니다 근육 섬유에는 EBD 형광을 보여주는 (A) 야생형 형제.. (B) 여러 근육 섬유 (화살표) 내 EBD 형광와 Sapje ​​동형 접합 돌연변이. 모든 유충 EBD 주입 믹스 주입하고 3 DPF에서 4 시간 동안 배양 한 후에 분석 하였다. 형제 자매 및 돌연변이가 CCV 주입하기 전에 근육 섬유의 분리으로 분류되었다. <a href="https://www.jove.com/files/ftp_upload/53183/53183fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye at JoVE.com

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

에반스 블루 염료와 Zebrafish의 애벌레 골격 근육 무결성 분석

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

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JoVE Journal

Developmental Biology

9.0K 조회수.  The Hospital for Sick Children. In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

비디오: Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

시간 경과 브라이트 실체 현미경으로 Zebrafish의 애벌레에서 조직 복구를 캡처

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

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Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin &#38; Eosin staining that is widely used for subsequent morphological and morphometric analysis.

This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

Cardiotoxin 주입에 의한 급성 골격 근육 재생 유도

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning fundamental developmental biology through to large-scale genetic and chemical screens. However, many experiments, especially those related to infection and xenograft models, rely on microinjection and imaging of embryos and larvae, which are laborious techniques that require skill and expertise. To improve the precision and throughput of current microinjection techniques, we developed a series of microstructured devices to orient and stabilize zebrafish embryos at 2 days post fertilization (dpf) in ventral, dorsal, or lateral orientation prior to the procedure. To aid in the imaging of embryos, we also designed a simple device with channels that orient 4 zebrafish laterally in parallel against a glass cover slip. Together, the tools that we present here demonstrate the effectiveness of photolithographic approaches to generate useful devices for the optimization of zebrafish techniques.

Microinjection of zebrafish embryos and larvae is a crucial but challenging technique used in many zebrafish models. Here, we present a range of microscale tools to aid in the stabilization and orientation of zebrafish for both microinjection and imaging.

최적화 된 Microinjection 및 Zebrafish 애벌레의 이미징 microstructured 장치

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning ...

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

수축 성 활동 유도 된 쥐 골격 근육 Phenotypic 적응 연구 만성 자극에의 응용

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Pax7 Laminin 항 체와 면역 형광에 의해 골격 근육 인공위성 세포의 식별

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC&#160;expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.

골격 근 생검 절차에서 인간 근육 선조 세포의 격리, 배양, 특성화 및 분화

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this failure is the use of inadequate laboratory models. It is challenging to model complex human diseases in traditional laboratory organisms, but this issue can be circumvented through the study of human xenografts. The surgical method we describe here allows for the creation of human skeletal muscle xenografts, which can be used to model muscle disease and to carry out preclinical therapeutic testing. Under an Institutional Review Board (IRB)-approved protocol, skeletal muscle specimens are acquired from patients and then transplanted into NOD-Rag1null IL2r&gamma;null (NRG) host mice. These mice are ideal hosts for transplantation studies due to their inability to make mature lymphocytes and are thus unable to develop cell-mediated and humoral adaptive immune responses. Host mice are anaesthetized with isoflurane, and the mouse tibialis anterior and extensor digitorum longus muscles are removed. A piece of human muscle is then placed in the empty tibial compartment and sutured to the proximal and distal tendons of the peroneus longus muscle. The xenografted muscle is spontaneously vascularized and innervated by the mouse host, resulting in robustly regenerated human muscle that can serve as a model for preclinical studies.

Complex human diseases can be challenging to model in traditional laboratory model systems. Here, we describe a surgical approach to model human muscle disease through the transplantation of human skeletal muscle biopsies into immunodeficient mice.

면역 결핍 마우스에서 인간 골격 근 이종이식 수행

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this ...

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

마우스 골격 근 이식에서 기본 근구의 격리 및 분화

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

제브라피시 배아와 애벌레의 전체 마운트 면역항암화학

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, the muscle stem cell is activated and undergoes cell proliferation to generate a pool of myoblasts, which subsequently differentiate to form new muscle fibers. In many muscle wasting conditions, including muscular dystrophy and ageing, this process is impaired resulting in the inability of muscle to regenerate. The process of muscle regeneration in zebrafish is highly conserved with mammalian systems providing an excellent system to study muscle stem cell function and regeneration, in muscle wasting conditions such as muscular dystrophy. Here, we present a method to examine muscle regeneration in zebrafish models of muscle disease. The first step involves the use of a genotyping platform that allows the determination of the genotype of the larvae prior to eliciting an injury. Having determined the genotype, the muscle is injured using a needle stab, following which polarizing light microscopy is used to determine the extent of muscle regeneration. We therefore provide a high throughput pipeline which allows the examination of muscle regeneration in zebrafish models of muscle disease.

Skeletal muscle regeneration is driven by tissue resident muscle stem cells, which are impaired in many muscle diseases such as muscular dystrophy, and this results in the inability of muscle to regenerate. Here, we describe a protocol that allows the examination of muscle regeneration in zebrafish models of muscle disease.

근육 질환의 제브라피시 모델에서 근육 재생 검토

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, ...