우리는 zebrafish 축산 그의 도움 Beninio Jomok 주셔서 감사합니다. 이 작품은 NIH HL81753으로 후원됩니다.

1. 슬라이드의 준비 및 humidified 챔버 <ol><li> humidified 챔버는 비운 팁 박스 같은 상자에서 만들 수 있습니다. 챔버와 빛으로부터 샘플을 보호하기 위해 알루미늄 호일로 커버를 모두 넣어. </li><li> 챔버 내부, 건조에서 심장 샘플을 방지할 수 있습니다 챔버 내의 습도를 유지하기 위해 젖은 종이 수건 더미를 넣어. 슬라이드에 선반으로 종이 타월 위에 작은 microplate를 설정합니다. </li><li> 5x5 mm에 대한 차원과 심장 샘플 우물을 할 수있는 immEdge의 펜을 사용하여 현미경 슬라이드의 표면에 선 또는 원을 중 하나를 그립니다. 슬라이드가 건조하자. </li><li> humidified 챔버에 슬라이드를 넣고 각 우물에 50ul 신선한 4 % 포름 알데히드를 추가합니다. </li></ol> 2. 배아 심장의 해부 <ol><li> 생선 정지 몸 움직임까지 약 1 분 0.4 % tricaine을 포함 E3 계란 물 7 물고기의 배아를 마취 그래도 정상적인 마음을 가지고구타. </li><li> 해부 현미경의 무대에 오목 현미경 덮개를 씌우고. concaved 잘 수있는 배아를 전송합니다. </li><li> 명확하게 마음을 나타내기 위해 해부 현미경의 빛을 조합을 조정합니다. 개인 선호에 따라, 그것은 어두운 필드 또는 DIC과 같은 편광 상태 중 수 있습니다. </li><li> 다음 건조, 75 % 에탄올 두 개의 인슐린 주사기를 청소합니다. </li><li> 심장에 더 높은 배율과 집중 해부 현미경을 조정하십시오. 실제로 머리에 가까운 노른자 - 체절 경계 하나의 바늘 끝부분을 삽입하여 배아를 해결할 수 있습니다. 심장 지역에 다른 바늘 가까이를 삽입하고 신속하게 심장을 가져옵니다. 심장이 완전히 배아로부터 분리되지 않을 경우 심장과 노른자 사이에 남아있는 연결 조직을 잘라. </li><li> 분리된 심장에 낮은 배율과 초점 현미경을 조정합니다. 10μl pipetteman을 사용하여 1μl 볼륨으로 설정합니다. 중 그리거나 만지지로 pipetteman의 팁을 적용격리된 심장 내에서 또는 팁의 표면에 부착하거나되도록 심장 샘플. </li><li> 준비된 슬라이드에 4 % 포름 알데히드 용액에 피펫 팁을 찍어 및 솔루션에 마음을 공개하는 피펫 피스톤을 놓으면 되죠. 물 표면의 장력은 또한 피펫 팁에서 솔루션 마음을 출시하는 데 도움이됩니다. 각 잘 3 마음보다 놔. </li></ol> 3. Immunostaining <ol><li> 챔버를 닫고 부드러운 출렁 이는 운동과 함께 흔드는에 넣어. 상온에서 20 분 심장 샘플을 수정. </li><li> 슬라이드의 뒷면에있는 물을 닦아 및 교환 버퍼에 대한 해부 현미경의 무대에 슬라이드를 넣어. </li><li> 각 우물에 빈 영역에 10μl pipetteman에서 팁을 딥과 심장 표본에서 최대한 팁을 유지. 솔루션 끝에 그려진 때 마음은 물 흐름과 함께 이동 수도 있기 때문에, 팁에 연결 마음을 피하기 위해주의를 가져가라. 변화필요한 경우 팁 현지화. </li><li> 조직에 솔루션을 폐기하고 심장 샘플 쉽게 기포의 존재에 손실이기 때문에 끝에 거품을 생산하지 마십시오. 짧은 시간 동안 심장 샘플 부분적으로 건조 유지하면 마음이 슬라이드에 부착된 계속 도움이 될 수 있습니다. </li><li> 심장 샘플을 커버 5 분 느린 출렁 이는 운동의 humidified 챔버에서 부화 50μl PBST를 추가합니다. 한 번 더 씻어 반복합니다. 이 단계 후, 심장 샘플 4 humidified 챔버 최대 일주 ° C.에 저장될 수 있습니다 </li><li> 상온에서 1 시간 PBST에서 10 % 양의 혈청과 심장 샘플을 차단합니다. </li><li> 이러한 β - catenin 항체의 1:200 희석 및 mef2 특정 항체의 1:200 희석과 같은 기본 항체와 심장 샘플을 품어, 10 % 양의 실온에서 1 시간 동안 혈청 / PBST. </li><li> 상온에서 PBST 세 번 (5 분. 각)과 심장 샘플을 씻으십시오. </li><li> 보조로 샘플을 품어PBST에서 0.5 시간 동안 이러한 알렉사 - 태그 안티 - 마우스 및 / 또는 방지 토끼 항체의 1:1000 희석으로 항체,. </li><li> PBST로 세 번 심장 샘플을 씻으 (5 분. 각) 실온에서합니다. </li></ol> 4. 이미징 <ol><li> PBST를 제거하고 각 잘 ~ 10 μl 장착 매체를 추가합니다. 솔루션은 분명해진다 때까지 몇 분 동안 곳에는 바위. </li><li> 장착 매체의 대부분을 제거하고 현미경 커버 유리 (24x50) 한 슬라이드에있는 우물을 모두 커버. </li><li> 자이스 혈구 Axioplan이 현미경을 사용하여 이미지 물고기 마음이 ApoTome (칼 자이스 혈구)를 갖추고 있습니다. </li></ol> 5. 대표 결과 모든 zebrafish의 cardiomyocytes의 막과 핵을 드러 이미지의 예제는 그림에 표시됩니다. 1. mef2c 및 β - catenin 항체는 52 hpf의 zebrafish 배아에서 심장 해부 샘플을 얼룩 함께 사용되었습니다. 동안 mef2c 항체 얼룩 cardiomyocytes의 핵, β - catenin항체는 각 세포 8-10의 경계를 나타낸다. 복합 현미경의 무대를 조절함으로써, 심장 샘플 이미지는 외부 곡률과 마음 11 내부 곡률 관찰 수 같은 방향으로 조정할 수 있습니다. 총 cardiomyocyte 번호, cardiomyocyte 세포 크기, 순환성 그때 측정할 수 있습니다. 배아 심장의 구조를 보여줍니다 sarcomeric 또 다른 예제는 그림에 표시됩니다. 2. 우리는 해부 배아 마음에 F59, 마이 오신의 항체를 사용 immunostaining 수행했습니다. 두꺼운 필라멘트의 줄무늬 밴드가 높은 배율 (그림 2) 6 계시 수 있지만 전체 배아 중심에 myofibril 네트워크는 명확하게, 낮은 배율에서 발표하실 수 있습니다. <img alt="그림 1" src="/files/ftp_upload/3510/3510fig1.jpg" /> 그림 1. 핵과 나와!를 표시 mef2 (적색)과 β - catenin (녹색)의 Immunostainingzebrafish의 뇌실에서 cardiomyocytes의 아이 네스. 스케일 바 = 10μm <img alt="그림 2" src="/files/ftp_upload/3510/3510fig2.jpg" /> 그림 2.하거나 낮은 배율 (A) 또는 높은 배율 (B)에 zebrafish 뇌실에서 두꺼운 필라멘트를 보여 마이 오신의 Immnostaining. 규모 막대 = 10μm

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관심 없음 충돌 선언하지 않습니다.

고전 온 마운트 immunostaining 방법에 비해, 우리의 방법은 다음과 같은 장점이있다. 첫째, 훨씬 강한 형광 신호를 지속적으로 개선 침투력으로 인해 얻을 수 있습니다. 전체 마운트 immunostaining 방법에서 마음을 둘러싼 치밀한 피부 조직이 상당히 전신 높은 배경의 결과로, 많은 항체의 침투를 감소. 이 문제는 3 일 후 수정 (dpf)보다 오래된 배아에 특히 심각합니다. 반대로, 해부하는 마음은 훨씬 더 침...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> tricaine </td><td> 연구 유기물 </td><td> 3007T </td></tr><tr><td> 포름 알데히드 </td><td> Polysciences </td><td> 04,018 </td></tr><tr><td> 인슐린 주사기 </td><td> Becton 디킨슨 </td><td> 329,461 </td></tr><tr><td> 트리톤 - X - 100 </td><td> 시그마 </td><td> T8532 </td></tr><tr><td> 안티 β - catenin 항체 </td><td> 시그마 </td><td> C7207 </td></tr><tr><td> 안티 Mef2c 항체 </td><td> 산타 크루즈 생명 공학 </td><td> SC313 </td></tr><tr><td> F59 </td><td> Zfin </td><td></td></tr><tr><td> 안티 α - actinin 항체 </td><td> 시그마 </td><td> A7811 </td></tr><tr><td> 안티 Ilk 항체 </td><td> 세포 신호 </td><td> # 3862 </td></tr><tr><td> 알렉사 형석 568 염소 안티 토끼 IgG </<html> TD&gt; <td> Invitrogen </td><td> A11011 </td></tr><tr><td> 알렉사 형석 488 염소 안티 마우스 IgG1 </td><td> Invitrogen </td><td> A21121 </td></tr><tr><td> 형광 장착 매체 </td><td> 벡터 </td><td> H - 1200 </td></tr><tr><td> ImmEdge 펜 </td><td> 벡터 </td><td> H - 4000 </td></tr><tr><td> 폴리 - L - lysin 코팅 슬라이드 </td><td> 전자 현미경 과학 </td><td> 63410-01 </td></tr><tr><td> 현미경 덮개 유리 </td><td> 어부 </td><td> 12-543 - D </td></tr><tr><td> Concaved 현미경 슬라이드 </td><td> 어부 </td><td> 7-1305-8 </td></tr><tr><td> 해부 현미경 </td><td> Leica MZ95 </td><td></td></tr><tr><td> 복합 현미경 </td><td> 자이스 혈구 </td><td> Axioplan2 </td></tr><tr><td> ApoTome </td><td> 자이스 혈구 </td><td></td></tr></tbody></table> PBST : 1 X PBS 0.5 % 트리톤 X - 100 엔트 &quot;&gt; 25X Tricaine : 400 밀리그램 Tricaine 97.9 ML ddH 2 O 2.1 ML (1M 트리스 pH9) 7.0 산도를 조정

immunostaining of dissected zebrafish embryonic heart

Zebrafish의 배아는 심장 개발과 유리 발생학 및 유전학 1,2으로 인해 인간의 심장 질환을 공부에 대한 생체내의 척추 모델의 인기가됩니다. 약 100-200 배아는 성인 물고기 한 쌍의에서 매주 쉽게 사용할 수 있습니다. 예 utero을 개발 투명 태아는 심장 결함에게 3 평가에 이상적합니다. 어떤 유전자의 표현은 morpholino 기술 또는 RNA 사출 사를 통해 조작하실 수 있습니다. 또한, 앞으로 유전자 스크린은 이미 cardiogenesis 5 가지 관점에 영향을 미칠 돌연변이의 목록을 생성합니다. 전체 마운트 immunostaining는 특정 조직 6 타겟 단백질의 표현 패턴을 나타내기 위해이 동물 모델에서 중요한 기술입니다. 그러나, 세포 또는 subcellular 구조를 확인할 수 있습니다 고해상도 이미지는 실제 locat으로 인해 주로 어려운되었습니다마음과 항체의 가난한 들어간 이온. 여기, 우리가 먼저 마음을 해부 다음 현미경 슬라이드의 표면에 얼룩 과정을 실시하여 이러한 병목 현상을 해결하기 위해 방법을 제시한다. 작은 마음이 샘플의 손실을 방지하기 위해 및 솔루션 처리를 촉진하기 위해, 우리는 immEdge 펜으로 그려진 현미경의 표면 슬라이드에 동그라미 안에 심장 샘플을 제한. 얼룩 후, 형광 신호를 직접 복합 현미경으로 볼 수 있습니다. 배아 물고기의 마음은 몇 가지 세포 레이어로 구성되어부터 새로운 방법, 항체에 대한 침투를 상당히 향상시킵니다. 그대로 마음의 고품질 이미지는 일 2 일 6 세 zebrafish 배아에 대해 훨씬 감소 행렬 시간 내에 얻을 수 있습니다. 우리의 방법은 잠재적으로 zebrafish이나 다른 작은 동물 중 하나에서 해부하는 다른 장기를 얼룩이 확장될 수 있습니다.

Watch this Scientific Journal Video about Immunostaining of Dissected Zebrafish Embryonic Heart at JoVE.com

Immunostaining of Dissected Zebrafish Embryonic Heart

zebrafish 배아 심장의 immunostaining를 수행하는 빠른 방법이 설명되어 있습니다. 전체 마운트 immunostaining 접근 방식에 비해,이 방법은 크게 크게 감소 처리 시간 이내에 심장에 세포 / subcellular 구조를 공개 고해상도 이미지를 얻는 수있는 항체의 침투를 증가시킵니다.

해부 Zebrafish 배아 심장의 Immunostaining

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

immunostaining-of-dissected-zebrafish-embryonic-heart

Research

JoVE Journal

Biology

17.5K 조회수.  Mayo Clinic College of Medicine. zebrafish 배아 심장의 immunostaining를 수행하는 빠른 방법이 설명되어 있습니다. 전체 마운트 immunostaining 접근 방식에 비해,이 방법은 크게 크게 감소 처리 시간 이내에 심장에 세포 / subcellular 구조를 공개 고해상도 이미지를 얻는 수있는 항체의 침투를 증가시킵니다.

비디오: Immunostaining of Dissected Zebrafish Embryonic Heart

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

공촛점 현미경으로 Zebrafish 배아 뇌 영상 라이브

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

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Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

조직 타겟 배아 Chimeras : Zebrafish Gastrula 세포 이식

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

의 전자 현미경을위한 배아의 준비 Drosophila 태아의 심장 관

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Microdissection of Zebrafish Embryonic Eye Tissues

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post fertilization (dpf), the larvae will show the first visual response. Many genes have been identified to control a proper eye development, but we are far from a complete understanding of the underlying genetic architecture. Whole genome gene expression profiling is a useful tool to elucidate genetic regulatory network for eye development. However, the small size of the embryonic eye in zebrafish makes it challenging to obtain intact and pure eye tissues for expression analysis. For example, the anterior-posterior length of the eye between day 2 and 3 is only approximately 200-300 &mu;m, while the diameter of the lens is less 100 &mu;m. Also, the retinal pigment epithelium (RPE) underlying the retina is just a single-layer epithelium. While gene expression profiles can be obtained from the whole embryo, they do not accurately represent the expression of these tissues. Therefore pure tissue must be obtained for a successful gene expression profiling of eye development. To address this issue, we have developed an approach to microdissect intact retina and retina with RPE attached from 1-3 dpf, which cover major stages of eye morphogenesis. All procedures can be done with fine forceps and general laboratory supplies under standard stereomicroscopes. For retinal dissection, the single-layer RPE is removed and peeled off by brushing action and the preferential adherence of the RPE remnants to the surface of the culture plate for dissection. For RPE-attached retinal dissection, the adherence of RPE to the dissection plate is removed before the dissection so that the RPE can be completely preserved with the retina. A careful lifting action of this tissue can efficiently separate the presumptive choroid and sclera. The lens can be removed in both cases by a chemically etched tungsten needle. In short, our approach can obtain intact eye tissues and has been successfully utilized to study tissue-specific expression profiles of zebrafish retina1, 2 and retinal pigment epithelium3.

This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos.

Zebrafish 배아 눈 조직의 Microdissection

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

배아 림브 스킨 Vasculature에 대한 전체 마운트 Immunohistochemical 분석 : 엠브료에 혈관 분기 Morphogenesis를 공부하는 모델 시스템

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

애벌레의 심장 해부, 소년과 성인 Zebrafish, Danio rerio</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

위한 시스템<em&gt; 예 생체</em배아 췌장의&gt; 배양

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

Zebrafish 배아 골격근의 레이저 쏜 부상

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 &mu;m) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.

Zebrafish are an emerging system for modeling human disorders of the skeletal muscle. We describe a fast and efficient method to isolate skeletal muscle myofibers from embryonic and larval zebrafish. This method yields a high-density myofiber preparation suitable for study of single skeletal muscle fiber morphology, protein subcellular localization, and muscle physiology.

해리 된 준비에서 배아와 애벌레 Zebrafish의 골격 근섬유의 분석

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

소설<em&gt; 생체</em배아 마우스 사랑을&gt; 문화 방법

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...