这些方法是由鳗鱼在JT的实验室支持DePaul大学研究委员会和美国国立卫生研究院/ NIDCR R01授予JT（DE016678）提供的研究休假期间。我们非常感谢加猎人，在CMRC斑马鱼核心设施，动物保健技术人员和宝琳娜Pawluczuk，我们的本科实验室助理的努力。

斑马鱼的饲养斑马鱼是按1标准方法安置和饲养。过去的后期幼虫时期，一个斑马鱼的发育阶段可以衡量它的标准长度（SL），或从上唇anteriormost的角度以毫米为单位的直线距离尾鳍（posteriormost hypural板）的基础2。在受精后约一个月（SL 10-12毫米），鼻和上颌骨的触须出现嗅觉坑附近的颚后的角落，分别为透明上皮芽。由于斑马鱼的成熟，触须延伸到狭窄，晶须状的附加物。由于上颌barbel较大（2-3毫米），更易于操作，我们的协议只适用于这个特定的附属物;然而，类似的技术，可适应较小的鼻barbel。 Barbel裁剪<ol><li>在所需的阶段（例如，1.5-2.5厘米SL，〜3-6个月后受精）麻醉成年斑马鱼浸泡在0.015％的MS - 222（乙基氨基磺酸）缓冲系统水的pH值7.0。和鳃通风变得缓慢和定期观察，直到停止游泳运动。根据鱼的大小，完成麻醉大约需要2-5分钟。 </li><li>使用水族馆渔网，一次传输一个在培养皿中的湿纸巾折叠1-3鱼。使用钝的铲，东方的鱼，所以他们是平行的相互左外侧的一面向上。在覆盖了整个身体的鳃盖鱼（鳃盖）在手术过程中保持湿润的皮肤和鳃尾部分湿纸巾的褶皱部分。 </li><li>体视显微镜下的Petri板，采用低角度的入射光照射头部区域。专注于左上颌窦barbel，突出从上颌骨后腹角（图1）的基础。 <img src="/files/ftp_upload/1558/1558fig1.jpg" alt="图1" /> 图1。 </li><li>紧紧抓住的barbel颌骨边缘略有远端轴。提升barbel轴，并插入一个细尖的春天剪刀钳的近端的下颌。可触及的剪刀，镊子稳定。滑动轴的barbel剪刀的颌骨，直到尖端接近颌骨边缘。关闭剪刀划破barbel轴。截肢barbel，仍然笼罩在镊子，可用于固定或进一步的观察中删除。 </li><li>马上转移到一个干净的系统的水（约500毫升）含一滴亚甲蓝控制浅部真菌感染的小坦克鱼。让鱼一夜之间恢复。第二天早上，返回鱼的饲养系统。 Barbel再生是最大的，2周后。 </li></ol>琼脂嵌入匹配Barbel对<ol><li>通过适当的安乐死技术收集的鱼类和修复的4％，50毫升管多聚甲醛磷酸盐缓冲液（PBS）在4 ° C过夜搅拌。固定液的量应至少组织量的十倍。固定后，在核准的容器中的多聚甲醛废物处置和组织彻底冲洗PBS的几个变化。 </li><li>准备一个250毫升的玻璃烧瓶150毫升的2％琼脂糖（ 的 Tm〜37 ° C）蒸馏水。微波炉或热板加热，搅拌至完全溶解定期。在50-60℃水浴平衡的熔融解决方案。 </li><li>一个标准的凝胶电泳钻机组装放置一个小垫片胶的模具（10 × 10厘米〜100毫升凝胶体积）坚决反对的缓冲室的两侧。 2-4小齿样本的梳子（4 × 1.5毫米）。 </li><li>在一个水平面上，倒入模具融化的琼脂糖刚好盖住的梳子基地，形成很浅水井。立即把剩余的琼脂糖回温水浴，这将在以后使用嵌入组织（步骤9-12）。一个长长的玻璃巴斯德吸管放入烧瓶中，保持相同的温度琼脂糖的吸管。 </li><li>允许凝胶硬化20-30分钟。取出的梳子，然后删除包含从缓冲室，硬化凝胶的凝胶模具。坚决凝胶模具实验室磁带的磁带的开放两岸，使凝胶不会滑出。这盘录像带，应延长在琼脂糖表面几毫米，所以可以在以后添加额外的琼脂糖（13步）。 </li><li>立体显微镜下阶段的凝胶模具的位置。放大一个空井，并把关注焦点的边缘。 </li><li>上井相邻的琼脂糖表面，吸一滴的水或缓冲液固定barbel标本（ 例如，重新生成和控制）。 USI吴弯曲昆虫引脚，潮湿的标本，并将其推入井拖动底部。东方barbel轴相互平行，对井对面的长边对齐。表面张力，将有助于保持的组织中的地位。 </li><li>当准备嵌入，使用罚款枪头或删除多余的液体从井实验室组织的角落。工作很快，所以不要让组织干出来的。 </li><li>使用加热玻璃巴斯德吸管，轻轻吸液管和周围的标本新鲜熔化琼脂糖密封barbel组织。不要装得太满。琼脂糖变硬之前，与昆虫引脚的最后一分钟的调整。 </li><li>井旁边放置一个编号的纸张样本标签和安全用琼脂糖小降。 </li><li>每个标本以及重复步骤6至10。 </li><li>如果所需的图像校准，插入一张纸，印刷微米尺度（ 例如 <a href="http://incompetech.com/graphpaper/multiwidth/">，</a> http://incompetech.com/graphpaper/multiwidth/）到一个空井的 ​​底部。井底拼合的文件和温暖琼脂糖覆盖。 </li><li>所有的标本都嵌入后，将凝胶表面不规则，硬化滴琼脂糖。凝胶放置在一个平面上，轻轻地在融化的琼脂糖额外倒，直到上表面均匀。使用量最小的琼脂糖必要获得一个光滑的表面;太多琼脂糖晦涩的透射光摄影。允许此顶层变硬。 </li><li>凝胶可以被拍下立体显微镜记录每个barbel对大体形态的阶段。图像校准是通过拍摄嵌入式微米尺度。 </li><li>可以存储整个凝胶包裹在湿纸巾，并在几个星期在4 °塑料袋密封。 </li><li>作进一步的分析，可以对触须包含匹配的琼脂块切出的凝胶，用手术刀或刀片和石蜡组织学处理，或由3,4标准方法cryosectioning。另外，个体触须可以被解剖的琼脂糖细的针头，在水冲洗，和其他下游应用（ 例如，整个安装的免疫组织化学或激光共聚焦显微镜）处理。 </li></ol>

<ol>
	<li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+1+General+Methods+for+Zebrafish+Care.">Chapter 1 General Methods for Zebrafish Care.</a> The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Howe, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+length:+Not+quite+so+standard.">Standard length: Not quite so standard.</a> Fisheries Research (Amsterdam). 56, 1-7 (2002).</li><li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraffin+Embedding+Tissue+Samples+for+Sectioning.">Paraffin Embedding Tissue Samples for Sectioning.</a> Cold Spring Harbor Protocols. 6, (2008).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch+8.3+Agar+Embedding+for+Cryostat+Sectioning+of+Embryos+or+Larvae.">Ch 8.3 Agar Embedding for Cryostat Sectioning of Embryos or Larvae.</a> The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Hansen, A., Reutter, K., Zeiske, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taste+bud+development+in+the+zebrafish,+Danio+rerio.">Taste bud development in the zebrafish, Danio rerio.</a> Dev Dyn. 223, 483-496 (2002).</li></ol>

斑马鱼的上颌barbel是一个未充分利用的研究在斑马鱼的几种类型的细胞的生长，保养，并再生的组织系统。 barbel附属物虽然没有人类的模拟，它包含的细胞类型是高度保守的，从而有可能研究皮肤，腺体，黑色素细胞，循环系统血管和神经在光学明确和解剖学简单的圆柱形结构。 barbel组织类似的研究尾鳍，可诱发再生截肢。作为解剖标志的使用边界上颌骨，截肢平面，可以精确地放在促进barbel再生测量。对于一个有经验的运营商，每次手术只需要几秒钟。恢复是快速的，和我们迄今发现鱼的行为没有短期或长期的影响。一个上颌barbel游泳的斑马鱼，吃和繁殖作为有效的非手术控制，并具有可比性的生存组织收集的时间，最长6个月后手术。这种手术的生理影响的预测是微乎其微，因为：1）口外的口味进行芽的barbel还发现其他许多地方，鱼的上皮细胞，包括嘴唇，脸颊和头5， 2）与众不同的味蕾上出现在72小时内再生barbel（勒克莱尔等 ，未发表数据）。这使得上颌barbel微创系统，为研究伤口愈合，在成年脊椎动物中的心肌血运重建术，和再支配。 手术诱导再生后，触须可以收集的时间间隔形态测量和/或固定组织的微观分析。为方便起见，上颌barbel大约是一个斑马鱼胚胎的长度（2-3毫米）和直径（100-200毫米），促进应用的许多标准协议，包括石蜡组织学，cryosectioning，整个安装的免疫组织化学，和原位杂交。两者合计，这些特点使得上颌barbel高度在体内研究组织的修复和再生模式是可行的。

显微镜设备一个立体解剖显微镜与传输和事件光纤照明是需要进行barbel剪裁和琼脂嵌入。 Barbel裁剪<ul><li>鱼制水</li><li> 2斑马鱼渡坦克</li><li> MS - 222（乙酯3 - 氨基磺酸;西格玛化工＃E10521 - 10G） </li><li>无菌的120毫米Petri板</li><li>湿纸巾</li><li>钝性金属铲</li><li>杜蒙55＃生物Inox公司钳（精细的科学工具＃11255-20） </li><li>迷你Vannas弹簧剪刀（精细的科学工具＃15000-00） </li><li>亚甲蓝的抗真菌剂（Drs.福斯特史密斯＃CD - 905781） </li><li>小水族馆渔网</li></ul>组织收集和琼脂嵌入<ul><li>在1x 4％多聚甲醛磷酸盐缓冲液（PF - PBS），分装成50 ml锥形管和储存冷冻在-20 ° C，直到使用</li><li> 1X的磷酸盐缓冲生理盐水（pH值7.27-6） </li><li> 250毫升玻璃瓶中</li><li> 2％琼脂糖凝胶在蒸馏水</li><li>标准的小琼脂糖凝胶电泳仪（凝胶的大小=〜10厘米见方的） </li><li>小齿电泳的样品梳子（梳大小​​为4 × 1.5毫米） </li><li>温水浴（50-60℃） </li><li>实验室磁带</li><li>黑漆包线昆虫针，大小000（精细的科学工具＃26000-25） </li><li> 1-2针持有人（精细的科学工具＃26016-12） </li><li> 9英寸的玻璃巴斯德吸管</li><li>吸管灯泡或泵</li><li>实验室组织</li><li>小标签纸</li><li> （可选），印刷校准规模</li><li>湿纸巾</li><li>拉链封塑料袋</li><li>手术刀/刀片（切出琼脂块） </li></ul>

methods for the study of the zebrafish maxillary barbel

触须是在鱼类，爬行动物和两栖动物的皮肤感觉附属物。斑马鱼， 斑马鱼 ，开发两对触须，短鼻对一个较长的上颌对。 Barbel组织中含有的外胚层，中胚层和神经嵴起源的细胞，包括皮肤细胞，腺体，味蕾，黑色素细胞，循环系统血管及感觉神经。不像大多数成人组织，上颌barbel是透明的，让我们这些组织类型的开发和维护整个生命周期的可视化。 该视频显示上颌barbel（受精后约1个月开始）的早期发展，并演示了手术的协议，在成人的附属物（&gt; 3个月后施肥）诱导再生。简言之，麻醉鱼的左上颌窦barbel升高与远端尾椎上颌骨边缘的无菌镊子。罚款，无菌弹簧剪刀定位对镊子，在这一水平上削减barbel轴，建立了截肢平面解剖标志。这架飞机，并在比较对侧barbel蓄热式增长可测。 Barbel组织再生迅速，伤2个星期内达到最大的再生。 再生barbel分析技术包括解剖和嵌入在一个标准的DNA电泳井配对的触须（再生和控制）。方便嵌入式标本大体形态和形态学体视显微镜下拍摄的，可存储数周前，下游应用，如石蜡组织学，cryosectioning，和/或整装免疫组织化学。这些方法建立在体内组织系统内的斑马鱼的遗传背景下的多种类型的细胞的再生能力研究的新颖的上颌barbel。

Watch this Scientific Journal Video about Methods for the Study of the Zebrafish Maxillary Barbel at JoVE.com

Methods for the Study of the Zebrafish Maxillary Barbel

斑马鱼的上颌barbel外皮感觉器官含有外胚层，中胚层和神经嵴衍生物。更重要的是，成人barbel可再生后近端截肢。本视频介绍上颌barbel开发和演示了手术协议，barbel标本收集，嵌入和下游成像诱导再生。

上颌Barbel斑马鱼的研究方法

Barbels are skin sensory appendages found in fishes, reptiles and amphibians.  The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal ...

methods-for-the-study-of-the-zebrafish-maxillary-barbel

Research

JoVE Journal

Biology

12.4K 次观看.  DePaul University. 斑马鱼的上颌barbel外皮感觉器官含有外胚层，中胚层和神经嵴衍生物。更重要的是，成人barbel可再生后近端截肢。本视频介绍上颌barbel开发和演示了手术协议，barbel标本收集，嵌入和下游成像诱导再生。

视频: Methods for the Study of the Zebrafish Maxillary Barbel

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

计算机生成的动物模型的刺激

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

使用的视动反应研究斑马鱼的视觉功能

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

腹腔注射到成年斑马鱼

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

激光烧蚀斑马鱼Pronephros研究肾小管上皮再生

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

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 ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

再生的成年斑马鱼尾鳍在吗啉代入体内电穿孔

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

做自己动手恢复冻存样品的设备不慎掉落到低温储罐

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

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 ...

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to study the functions and evolution of bacterial symbionts in their host. As most symbionts cannot be cultured in vitro, methods to isolate an adequate quantity of bacteria for genome sequencing are very important. In the whitefly Bemisia tabaci, a number of endosymbionts have been identified and are predicted to be of importance in the development and reproduction of the pests through multiple approaches. However, the mechanism underpinning the associations remains largely unknown. The obstacle partially comes from the fact that the endosymbionts in whitefly, mostly restrained in bacteriocytes, are hard to separate from the host cells. Here we report a step-by-step protocol for the identification, extraction and purification of endosymbionts from the whitefly B. tabaci mainly by dissection and filtration. Endosymbiont samples prepared by this method, although still a mixture of different endosymbiont species, are suitable for subsequent genome sequencing and analysis of the possible roles of endosymbionts in B. tabaci. This method may also be used to isolate endosymbionts from other insects.

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Here, we present a protocol to isolate endosymbionts from the whitefly Bemisia tabaci through dissection and filtration. After amplification, the DNA samples are suitable for subsequent sequencing and study of the mutualism between endosymbionts and whitefly.

从白蛉提取内分泌物的方法<em&gt; Bemisia tabaci</em

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to ...