玛丽Birne，Areti Tsiola，Jaymie埃斯特韦斯和Arelys乌里韦感谢。这项工作是支持部分由纽约市立大学研究基金会和霍华德休斯医学研究所资助大学生的暑期计划（SPUR）和NIH RO3 41702-00-01。研究也支持由皇后学院和一些实验成像皇后学院，细胞和分子生物学的核心设施设备进行。

斑马鱼提出了使用标准协议，在皇后学院动物设施13在28 ° C 。鱼对或组交配应预制和清洗由此产生的胚胎，并在29 ° C培养箱中。鱼类的健康，应每天检查。在受精后5天（DPF），鱼应分为坦克在10-15鱼的密度和进入鱼设施育苗。这里介绍的协议，斑马鱼幼虫通过收集成人阶段：15，30，45，60，90，180 DPF。 1。斑马鱼的主题收集和固定<ol><li>卸下鱼栖息地槽，在0.2％Tricaine的净和地方麻醉。 </li><li>放置15分钟，安乐死在冰水中麻醉的鱼。 </li><li>在1x新鲜的4％多聚甲醛（PFA）磷酸盐缓冲液（PBS）。 </li><li>收集在4％煤灰的鱼在室温下1-2小时，然后过夜孵​​育4 ° C在培养皿封口膜包裹。这样可以使鱼的单位和以后清扫容易得多。 </li><li>固定后，在PBS冲洗两次吐温（PBT）。鱼可保持在4 ° C至10天，在PBT。 </li></ol> 2。鱼和解剖测量<ol><li>一条鱼放入培养皿的一半填充用新鲜PBS躺在其右侧。 </li><li>鱼类从吻端至尾基（尾柄）测量长度，不包括尾鳍，使用数字毫米卡尺（图1）。这是标准的长度（SL）。 </li><li>每条鱼可分配数字/字母的组合，让个别鱼类和导致心脏解剖，要为以后的分析跟踪。输入到电子表格中的数据，包括父母的库存数量，在固定，SL测量年龄，组织基于这些标签上。 </li><li>前清扫，标签PCR分配跟踪号码管带材和填充PBT或其他适当的解决方案作进一步的分析。 </li><li>东方鱼腹侧方在培养皿中，而稳定与身体之间的眼睛和鳃（图2）头钳。 </li></ol> 较小的鱼（大于12mm SL小）： <ol start="4"><li> a）使用尖锐镊子，或微针在针座，除去身体揭示心脏胸肌和鳍。在剥离过程中钳不断议案可能会导致移动的鱼在PBT的电流。特别是对于较小的鱼从而微针可以用来清除胸肌和鳍和打开体腔，这是问题。 </li><li>一）轻轻地使用镊子舀腔心脏从心房下。另外，在一些鱼，你可能会能够消除轻轻拉动球根出和心脏的其余部分将遵循的心脏。小心，如果使用这种技术，因为它很容易被破坏的中庭的位置。如果有额外的心脏，即罚款，并可以清除后的组织。 </li><li> a）如果鱼含有心脏一种荧光标记，如TG（myl7 GFP）的twu34，解剖使用荧光灯的范围，并确认切除心脏花期（图3）。 </li><li>一次）的心脏腔中删除，使用镊子举行的心脏和微针，消除心脏外额外的非心肌组织和心外膜衬砌。 </li></ol> 更大的鱼（大于12mm SL）： <ol start="4"><li> b）使用弹簧处理的微型剪刀，三不大于1mm深的切口：1 - 横向穿过腮2 - 横向切割前腹3 - 腹端连接都削减矢状切（图4）。 </li><li> b）使用尖锐镊子清除体内的胸肌和鳍打开体腔，并揭示了银色心包组织（图5A）。删除这个心包组织和心脏，将成为可见（图5B）。 </li><li> b）使用的微型剪刀切断连接到球根动脉，位于优越的心脏动脉。一旦动脉被切断，下庭钳提示，使用镊子舀腔心脏。如果有额外的心脏，即罚款的，可以删除后的组织。 </li><li> b）一旦心脏是从腔中删除，用两个镊子举行的心脏，并删除多余的组织，或一对镊子举行的心脏和微针，删除多余的，非心脏组织。心外膜衬砌，其黑色的色素沉着明显，可去除心脏外，如果需要，轻轻刮组织。 </li></ol> 3。摄影心中<ol><li>准备23克/ L，营养琼脂培养皿中，用PBS覆盖凝固的琼脂。 </li><li>使用镊子，在琼脂，使心脏在休息所需的方向，为拍摄好。 </li><li>祛瘀E的PCR管钳牢牢球根的一角，从心脏。将在琼脂培养皿的心脏。 </li><li>东方的心脏在琼脂照片。样本可旋转图片后，每到所有可能的方向（图6）。 </li><li>使用开销轻和短的曝光时间拍摄的心。轻质量的调整，如要获得最好的照片使用曝光。 </li></ol> 4。代表性的成果： 良好的清洁心脏解剖的一个例子是多个方向在图6所示。每个心脏有一定的差异，在整体形态，但心脏应该是完整的，包含一个完整的心室，心房和球根动脉。这种整体形态变化的一些例子，如图7所示。黑色斑点外膜组织，也可以从心脏中删除。 <img src="/files/ftp_upload/3165/3165fig1.jpg" alt="图1"/> 图1：测量从吻端至尾柄使用数字毫米卡尺的鱼。 <img src="/files/ftp_upload/3165/3165fig2.jpg" alt="图2"/> 图2。握住钳之间的眼睛和鳃鱼。 <img src="/files/ftp_upload/3165/3165fig3.jpg" alt="图3"/> 图3心脏特异荧光的转基因标记（如Tg 的 myl7：绿色荧光蛋白）。twu34，在这里显示，可以帮助在可视化较小的鱼，从解剖的心中。 <img src="/files/ftp_upload/3165/3165fig4.jpg" alt="图4"/> 图4三切口，去除胸鳍的肌肉和皮肤：1 -横向穿过腮2 -横向切割前腹3 -上矢状切开腹端连接都削减 。 <img src="/files/ftp_upload/3165/3165fig5.jpg" alt="图5"/> 图5：黑色字母标签的心脏腔：球根动脉（BA），心室（V），和庭（一）。 一个上身腔是开放的，以显示心包银色组织。 B超取出后心包，心脏是可见的，容易去除。 <img src="/files/ftp_upload/3165/3165fig6.jpg" alt="图6"/> 显示的心脏图6。不同的方向。黑色字母标签的心脏腔：球根动脉（BA），心室（V），和庭（一）。灰色箭头表示参考其在体内的正常位置的心脏的位置和方向：（一）前，后（P），背（四），腹（五），左（L）和右（R）。比例尺表示400μm。 一个心脏的腹面观 B超背的心脏 从左侧的C -侧视 从右侧的D -侧视 <img src="/files/ftp_upload/3165/3165fig7.jpg" alt="图7"/> 图7，不同大小的心的形态显示。黑色字母标签的心脏腔：球根动脉（BA），心室（V），和庭（一）。比例尺表示400微米。 AC心SL形式的鱼不大于12mm DF -红桃SL形成鱼大于12mm

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	<li>Ahmad, N., Long, S., Rebagliati, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+southpaw+joins+the+roster:+the+role+of+the+zebrafish+nodal-related+gene+southpaw+in+cardiac+LR+asymmetry.">A southpaw joins the roster: the role of the zebrafish nodal-related gene southpaw in cardiac LR asymmetry.</a> Trends Cardiovasc Med. 14, 43-49 (2004).</li><li>Armstrong, E. J., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+valve+development:+endothelial+cell+signaling+and+differentiation.">Heart valve development: endothelial cell signaling and differentiation.</a> Circ Res. 95, 459-470 (2004).</li><li>Bakkers, J., Verhoeven, M. 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没有利益冲突的声明。

后胚胎鱼斑马鱼的心脏解剖这些方法使一个完好的心脏彻底清除。虽然这种技术很简单，并提供外部的削减，这将防止心脏损害的指导，一个稳定的手较小的鱼。 在夹层中的一个关键步骤是识别银色心包，心脏是直接在此囊。心包可视化提供了一个寻找和最终消除心脏形式腔体内的重要标志。此外，切割优于球根动脉的动脉将防止其他心脏的球根无意搬迁。在较大的鱼，它?...

<table border="1"><tbody><tr><th>材料</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>多聚甲醛</td><td>电解二氧化锰</td><td> PX0055 </td><td> .04g/mL在PBS </td></tr><tr><td>钳＃55 </td><td>福斯特</td><td> 11295-51 </td><td></td></tr><tr><td>微型剪刀</td><td>福斯特</td><td> 15005-08 </td><td></td></tr><tr><td>针座</td><td>福斯特</td><td> 26016-12 </td><td></td></tr><tr><td>微针</td><td>福斯特</td><td> 10130-10 </td><td></td></tr><tr><td>营养琼脂</td><td> BBL </td><td> 11472 </td><td>在蒸馏水中H 2 O23克/大号</td></tr><tr><td>培养皿</td><td>屋宇署猎鹰</td><td> 351029 </td><td></td></tr><tr><td> PCR扩增8管带</td><td>美国科学</td><td> 2502至00年</td><td></td></tr><tr><td>数显卡尺</td><td> Fisher Scientific则</td><td> 14-648-17 </td><td></td></tr></tbody></table>

heart dissection in larval, juvenile and adult zebrafish, danio rerio

斑马鱼已成为一种有益和实用的模式生物，胚胎心脏发育的研究 （见1-6最近的评论），但是，工作研究通过成人心脏发育的胚胎后一直有限7-10。研究改变形态的成熟和老化的心脏被限制缺乏技术可用于分期和隔离，少年和成人的心。为了分析对鱼的寿命心脏的发展，我们在许多阶段剖析斑马鱼的心和照片作进一步的分析 11 。心脏的形态特征，可以很容易地被量化和个人的心，可以进一步分析的标准方法。斑马鱼生长在浮动利率和成熟的相关性更好，鱼比年龄大小，因此，固定后，我们作为衡量鱼成熟的鱼照片和测量长度。该协议解释两个不同的，对斑马鱼的大小而定清扫技术，从幼虫3.5mm标准长度（SL）不等，心中100μm的心室长度（VL），成年人，SL为30mm和VL1毫米或更大。幼虫和成鱼有相当不同的身体和器官形态。幼虫不仅明显变小，他们较少有色素和各器官的是视觉上很难辨别。出于这个原因，我们使用不同的夹层技术。 我们用解剖前的固定程序，我们发现，解剖后，euthanization直接的心中有一个变数较多的形态很松散，像气球心房相比，删除以下固定的心中。鱼固定剥离之前，保留在体内的形态和商会位置（数据未显示） 。此外，用于演示目的，我们采取的心脏（心肌）的具体GFP转基因TG（myl7 GFP）的优势twu34（12），这使我们看到整个心脏，并在早期阶段，特别是在发展有用的心脏时，形态是从周围组织明显。解剖的心脏原位杂交，免疫组织化学，RNA提取或其他分析方法在后胚胎斑马鱼更容易在心脏的发展和成熟使用的基本细胞和分子生物学的进一步分析。该协议将提供一个有价值的技术心脏发育成熟和老化的研究。

Watch this Scientific Journal Video about Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio at JoVE.com

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

一个明确的，标准化的方法是在多个发育阶段的斑马鱼的心脏解剖和隔离。注释和定量分析技术进行了讨论。

幼虫，少年和成年斑马鱼的心脏解剖，斑马鱼</em

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

heart-dissection-in-larval-juvenile-and-adult-zebrafish-danio-rerio

Research

JoVE Journal

Biology

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

21.3K 次观看.  Queens College, City University of New York. 一个明确的，标准化的方法是在多个发育阶段的斑马鱼的心脏解剖和隔离。注释和定量分析技术进行了讨论。

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

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

在果蝇幼虫中枢神经系统的剖析

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

科研

生物学

Drosophila Larval NMJ Dissection

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 35 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. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

果蝇幼虫NMJ夹层

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

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

格罗斯和精细解剖内耳感觉上皮细胞在成年斑马鱼（斑马鱼）

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

剖析器官的成年斑马鱼

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

夹层和染色果蝇幼虫卵巢

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

成年斑马鱼肾脏的解剖

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

在体内电穿孔Morpholinos到成年斑马鱼视网膜

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.

Regular Care and Maintenance of a Zebrafish Danio rerio Laboratory: An Introduction

This protocol outlines regular maintenance and care to maintain optimal conditions for zebrafish husbandry. The video illustrates the protocol for system maintenance, regular housing, feeding, breeding, and raising of zebrafish larvae.

定期保养和维护的斑马鱼（<em&gt;斑马鱼</em&gt;）实验室简介

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

在体内病态的人类基因组建模使用斑马鱼

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavaging成年斑马鱼

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...