The authors thank Meng-Chieh Shen for images, Jennifer Anderson for providing helpful comments on the manuscript, and members of the Farber laboratory for their contributions in developing these techniques. This study was funded by NIDDK-NIH award RO1DK093399 (S.A.F.), RO1GM63904 (The Zebrafish Functional Genomics Consortium: PI Stephen Ekker and Co-PI S.A.F), and F32DK096786 (J.P.O.). This content is solely the responsibility of the authors and does not necessarily represent the official views of NIH. Additional support was provided by the G. Harold and Leila Y. Mathers Charitable Foundation to the laboratory of S.A.F and the Carnegie Institution for Science endowment.

这些协议已批准由卡内基科学研究所机构动物护理和使用委员会（协议编号139）。 1.动物的制备<ol><li>保持在14小时成虫和幼虫在28°C:10小时亮:暗周期。每日两次与壳牌免费卤虫 （拆封，不孵化，开始在14 DPF）和商业微丸饲料成年人。 </li><li>这些协议的使用6-7 DPF幼虫由AB背景的自然产卵收集优化。协议可以修改为其他年龄和背景的幼虫。不提供外源性食物前6 DPF。 </li><li>麻醉与三卡因在胚胎介质（EM）的幼虫（4.2毫升4毫克每100毫升的EM / ml的三卡因）在室温（RT）。 </li></ol> 2.富含脂质蛋黄饲料的研制<ol><li>准备和储存鸡蛋蛋黄。分开12鸡蛋蛋黄和蛋白。池蛋黄，等分1毫升到1.5毫升管，并储存在-80°C至1年（12蛋黄会让〜80等分）。池白人，存储和脂质差，富含蛋白质的饲料使用。 </li><li>制备荧光脂质类似物（多个）如果需要的话也可以加入到蛋黄饲料。 <ol><li>根据制造商的说明暂停购买的，粉末状的荧光脂质在100％乙醇或100％氯仿类似物在0.1微克/微升（见下面的注释讨论溶剂），等分试样在不透明玻管，用石蜡膜密封，并存储。由于有机溶剂的迅速蒸发不使用等份体积小于〜400μl的用于长期存储。 注意:如果所述脂质类似物悬浮在乙醇中，脂质类似物的大量将被使用，因为它在 N 2下干燥快于氯仿。如果脂质类似物悬浮在乙醇它不具有在步骤2.2.4它添加到脂质体进料之前被干燥并重新悬浮，但总乙醇浓度不应超过0。在脂质体饲料1％以避免乙醇潜在的混杂生理效应。 </li><li>荧光脂肪酸类似物:准备在EM20毫升5％蛋黄乳剂的总体积。从该制备每份5毫升与6.4μM4,4-二氟-4-硼杂3α，4α二氮杂-s-苯并二茚的实时共焦成像和食物摄取测定或1μg/ ml的终浓度（BODIPY C16） BODIPY C 12的立体成像。转移荧光脂质类似物的所需量到1.5毫升的塑料管中。干脂质下N 2流，注意不要吹出来脂管。 </li><li>随即，在悬浮移液器流下来管的两侧5微升100％的乙醇，加入95微升EM，拌匀。 注:该混合物应该出现均匀;如果彩色脂残留在管的侧面或液体出现块状，脂质还没有完全溶解，并且需要更多的乙醇。保护管来回m个光并存储在冰上。 </li><li>荧光胆固醇类似物:在5ml 0.5-5％蛋黄乳剂实时成像研究制备2.4微克/毫升胆固醇的最终浓度。转移胆固醇所需量到1.5毫升的塑料管中。干脂质下N 2流，注意不要吹出来脂管。 </li><li>立即，重悬在15微升的RT的100％乙醇吹打流走在管的两侧，并在纯化H 2 O与85微升的RT 1％无脂肪酸的BSA混合避光管并存储在冰上。 </li></ol></li><li>准备蛋黄脂质体饲料。 <ol><li>蛋黄的解冻等分试样添加到EM在50毫升锥形管中。涡稀释蛋黄混合物1-2分钟;该混合物应该似乎是均匀的乳液。 </li><li>通过细滤网倒入混合液，以去除蛋白质集团，并收集在一个新的，新鲜的50毫升锥形管。 </li><li>脉冲sonica在室温下创建的脂质体:;忒蛋液用四分之一英寸的锥形微尖（6 W输出强度的5倍（5×1秒，停1秒关闭）暂停各组间间隔5-10秒）。超声器电源设置仪器依赖，因此需要每个仪器和微尖的优化。 注:在室温连续晃动脂质体混合物，以保持均匀待用。 </li></ol></li><li>荧光脂质类似物添加到蛋黄脂质体，如果需要的话。超声处理后，立即吸管调制脂质到脂质体混合物和涡旋以高速进行30秒掺入到脂质体中。通过包装箔管避光混合物，并继续在室温下摇摆。 </li></ol> 3.饲养范式<ol><li>准备幼虫喂养。 <ol><li>在此之前开始进料，屏幕幼虫可能妨碍喂食明显的形态缺陷。转移幼虫60毫米×15毫米的培养皿或6-或12孔PLATES，EM降低到最低限度，并用5毫升制备的脂质体溶液的更换。 </li><li>如果需要的话，未喂食对照幼虫转移至5ml EM和/或5毫升5％蛋清在EM和并行处理。 </li></ol></li><li>轻轻摇动，在培养摇杆幼虫（30转29-31°C）在整个饲料，鼓励食物的摄入量，同时继续从光盾，如果荧光脂类似物都存在。如果幼虫需要被暴露于光，使与一个有色玻璃培养箱罩曝光。 </li><li>在饲养期结束时，冲洗自由游泳的幼虫在EM中的3倍。 注:幼虫开始在饲料中的任何一点消耗脂质体。如果这是至关重要的幼虫开始在同一时间喂养，屏幕食物的摄入量（见3.4）后，饲养1小时，只返回已开始喂养蛋黄乳液来完成饲料的幼虫。 </li><li>屏幕在这一点食物摄入幼虫少数不得EAT（一般0-5〜％）。审查已麻醉或略微在冰上冷却的金属块上，以确定他们是否供给幼虫的肠中:已消耗脂质体幼虫将具有变暗肠（ 图1）。 </li><li>图片或过程幼虫立即食物的摄入。或者，在新鲜的EM地方幼虫，并在28℃下孵育以允许发生代谢的处理。 </li></ol> 4.安装幼虫实时成像<ol><li>准备安装介质。 <ol><li>为3％的甲基纤维素:添加0.75克甲基纤维素至25ml接近沸腾EM，旋涡，岩石在RT至完全溶解2-3天，并储存在4℃的多年来在-20℃。在4℃下冷冻和解冻的重复循环将去除气泡。在刨冰店3％甲基纤维素而安装的幼虫。 </li><li>对于1.2％的低贴装琼脂糖:添加将0.3g琼脂糖低熔点至25毫升EM和通过将煮沸，分装于2ml溶解ŧubes，并在散热块上26-28℃的维护。未使用的等分试样可被存储在RT或-20℃，并在使用时再次煮沸。确保琼脂糖是不够冷静暴露于幼虫，也可能是过热之前来处理。 </li></ol></li><li>对于低倍率，介质通量实时成像，放置在冰上的金属块上的组织载玻片并等待滑动冷却。粘在应用3％甲基纤维素大方量（已保持在4℃的冰）到幻灯片，转移幼虫到3％甲基纤维素，使用钓线扑克（0.41毫米直径的渔线玻璃毛细管的端部）创建一个誓言排出过量的EM（使甲基纤维素不会稀释后）和根据需要的幼虫放置。 </li><li>对于短期（&lt;20分钟），以正直的油再现客观的高倍率实时成像，支座与盖玻片瘦到方法幼虫。 <ol><li>使用基于氰基丙烯酸酯胶（强力胶），以附加氨基酸22毫米×30mm的盖玻片载玻片的一端。使用扑克把3％的甲基纤维素的细线上邻近于盖玻片的载玻片，并用大口径巴斯德吸管转移幼虫的幻灯片。删除多余的EM所以不会稀释甲基纤维素。 </li><li>使用扑克，轻轻的在他们的边甲基纤维素定位幼虫（左侧最高图像更加肝脏，右侧朝上图像胆囊）与他们的头旁边的盖玻片。 </li><li>在第二盖玻片的角，并轻轻点强力胶放置在安装盖玻片盖玻片和其他在幻灯片的一个边缘，桥接幼虫。注意不要在此步骤或同时成像，使幼虫仍然没有受伤客观上施加过大的压力。 </li></ol></li><li>对于长期，以正直的浸泡的目的高倍率成像，琼脂糖安装幼虫。幼虫添加到1.2％低熔点的一小等分试样的琼脂糖然后转移幼虫在小体积琼脂糖到培养皿。 <ol><li>迅速与琼脂糖凝固之前扑克定位幼虫。允许琼脂糖干燥2-3分钟，用新鲜的EM完全覆盖琼脂糖，以防止其干燥。 </li></ol></li><li>对于长期，有一个倒置的目标高倍率成像，安装在琼脂糖玻璃底菜幼虫。 <ol><li>在冰上冷却的金属块。将通过组织分开，以防止过度冷却和冻结幼虫金属块上的菜。转移幼虫的菜，并用纸巾吸液去除EM。 </li><li>将28°的幼虫顶部ç低熔点琼脂糖（1.2％）的下降，并立即用扑克（左侧到最佳图像肝脏，右侧下到图像胆囊）位置。从冷块中移除盘，晾干2-3分钟，并添加新鲜的EM足够体积，以覆盖琼脂糖（〜2-5毫升）中。 </li></ol></li></ol> 5.食物摄入量化验<ol><li>在含有6.4微米的BODIPY C 16蛋黄脂质体饲料年底，在一个塑料1.5或2 ml管集中最低10幼虫洗净，去掉EM和捕捉冻结。样本可以被存储在避光几个月-80℃。 注意:如果可能的话，收集多个重复。它在此步骤以收集未喂食的幼虫正常化为背景脂质荧光（从同一离合器理想幼虫被使用）是重要的。 </li><li>执行修改后的布莱代尔脂质提取3,21。 <ol><li>将100μl均质缓冲液（20mM三Cl，1mM EDTA）中加入在冰上的样品（或者纯化H可使用2 O）。均质与微尖超声发生器冰（5秒共:1秒1秒关闭）。检查幼虫是完全均质;如果没有，重复。转移到13毫升一次性玻璃培养管（也可使用其它的玻璃管）。 注:氯仿​​是危险的;执行所有后续脂质前在RT在化学罩牵引步骤。 </li><li>对于每100微升匀浆缓冲液的使用时，添加375微升的1:2（氯仿:甲醇）。涡街30-60秒，孵化10分钟，加入125微升每使用100微升同质化缓冲氯仿，涡旋30秒。 </li><li>加200毫摩尔Tris pH为125微升7.5％用100微升匀浆缓冲液，涡旋30秒，离心（2,000×g下，5分钟，RT，避光）。小心地从离心分离机取出样品。它们将被分成两相:上部相是水性的，幼虫碎片的接口，以及一个较低的有机相（含有脂质）。 </li><li>用干净的玻璃吸管收集底部有机相，并将其转移到一个干净的13毫升玻璃管。 注:请注意，以避免在界面的水相和幼虫杂物;如果发生污染，重复离心步骤和追忆。丢弃上层，水相;它可能有助于移除并丢弃此收集有机相之前相。可将样品储存在-80℃下长达1个月。 </li><li>在真空下干燥有机相（0.12个大气压），同时避光。不要继续干样品后，液体蒸发，因为它会降低脂溶性。重悬脂质2:1（氯仿:甲醇），并存储在冰上。氯仿的最佳体积:甲醇溶液取决于所用的检测方法;开始低，继续稀释需要。一般原则是用10微升10池幼虫。 </li></ol></li><li>发现在均匀时间间隔对整个样品体积到引导TLC板上并扫描与常规用于生物分子成像（ 例如 ，荧光板读数器）的激光扫描仪的板。对于包括在具有500-650 nm和最大发射激发最大值从510-665纳米材料清单荧光脂类似物，使用488纳米的激光，并收集排放在520纳米。 <ol><li>量化与成像软件的每个点的总荧光。 注意:正常为通过减去配对，未喂食的幼虫样品的荧光天然存在的背景脂质荧光。 </li></ol></li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamental+approaches+to+the+study+of+zebrafish+nutrition.">Fundamental approaches to the study of zebrafish nutrition.</a> ILAR J. 53 (2), 144-160 (2012).</li><li>Farber, S. A., 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=Genetic+analysis+of+digestive+physiology+using+fluorescent+phospholipid+reporters.">Genetic analysis of digestive physiology using fluorescent phospholipid reporters.</a> Science. 292 (5520), 1385-1388 (2001).</li><li>Miyares, R. L., de Rezende, V. B., Farber, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+yolk+lipid+processing:+a+tractable+tool+for+the+study+of+vertebrate+lipid+transport+and+metabolism.">Zebrafish yolk lipid processing: a tractable tool for the study of vertebrate lipid transport and metabolism.</a> Dis Model Mech. 7 (7), 915-927 (2014).</li><li>Otis, J. P., 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=Zebrafish+as+a+model+for+apolipoprotein+biology:+comprehensive+expression+analysis+and+a+role+for+ApoA-IV+in+regulating+food+intake.">Zebrafish as a model for apolipoprotein biology: comprehensive expression analysis and a role for ApoA-IV in regulating food intake.</a> Dis Model Mech. 8 (3), 295-309 (2015).</li><li>Bligh, E., Dyer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+method+of+total+lipid+extraction+and+purification.">A rapid method of total lipid extraction and purification.</a> Can. J. Biochem. Physiol. 37, 911-918 (1959).</li><li>Otis, J. P., Farber, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+vertebrate+digestive+function+and+lipid+metabolism.">Imaging vertebrate digestive function and lipid metabolism.</a> Drug Discov Today Dis Models. 10 (1), (2013).</li><li>Andre, M., 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=Intestinal+fatty+acid+binding+protein+gene+expression+reveals+the+cephalocaudal+patterning+during+zebrafish+gut+morphogenesis.">Intestinal fatty acid binding protein gene expression reveals the cephalocaudal patterning during zebrafish gut morphogenesis.</a> Int J Dev Biol. 44 (2), 249-252 (2000).</li><li>Shimada, Y., Hirano, M., Nishimura, Y., Tanaka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+fluorescence-based+assay+system+for+appetite-regulating+gene+and+drug+screening.">A high-throughput fluorescence-based assay system for appetite-regulating gene and drug screening.</a> PLoS One. 7 (12), 52549 (2012).</li></ol>

The authors have nothing to disclose.

此处所描述的技术允许研究治疗幼虫斑马鱼用富脂质的饲料，在活幼虫可视膳食脂质加工，并量化幼虫的食物摄取。为确保成功，应特别注意考虑到几个关键步骤。商业鸡蛋不同;尽量减少潜在的可变性，我们执行的有机鸡蛋所有检测从没有被富集的Omega-3脂肪酸免费笼鸡。较低的投饲率可能在鱼类观察不到6岁DPF剩余卵黄内生的能源供应，不健康的鱼或鱼妨碍食物的摄入量（ 即下巴或肠畸形...

通过该肠调节膳食脂质加工机制，肝控制复合脂质合成和脂蛋白代谢和这些器官如何与中枢神经系统中工作，以控制食物摄取是不完全理解。它是生物医学兴趣的肥胖，心血管病，糖尿病和非酒精性脂肪肝疾病的当前流行的光来阐明这个生物学。在细胞培养研究和老鼠提供的大部分我们的膳食脂肪和疾病，和斑马鱼（ 斑马鱼 ）之间的机械关系的认识正在成为一种理想的模式，以配合这项工作。 斑马鱼也有类似的胃肠（GI）的器官，脂质代谢，脂蛋白运输到高等脊椎动物1,2，发展迅速，并在遗传上容易处理。幼虫斑马鱼的光学清晰度便于在体内研究中，particulař优势胃肠系统作为其胞外环境中的学习（ 即 ，胆汁，菌群，内分泌信令）几乎是不可能的模型离体进行。根据，研究相结合的遗传易处理性和有助文章的主体住斑马鱼的成像各种饮食控制（高脂肪3,4， -胆固醇5和-carbohydrate饮食6,7），与心血管疾病8的模型，糖尿病9,10，肝脂肪变性11-13，和肥胖14-16正在兴起，以提供代谢见解的主机。 过渡幼虫斑马鱼成代谢研究的一个重要方面是在其他动物模型的斑马鱼和利用斑马鱼的独特优势新颖测定的发展开发的技术的最优化。该协议提出的技术开发和优化的喂养幼虫斑马鱼梨皮D-一顿丰富的，可视化的整个身体饲料脂肪处理亚细胞分辨率，并测量食物的摄入。鸡蛋黄被选择以构成富含脂质的膳食，它包含高含量的脂肪和胆固醇（脂质组合〜鸡蛋黄，其中约5％的胆固醇，60％是甘油三酯，和35％的58％为磷脂的）。鸡蛋黄提供了比典型的商业斑马鱼微丸食物（〜15％的脂质）和优点，即它是与特定的脂肪酸种类的已知百分比的标准化供给更多的脂肪，如斑马鱼的饮食和喂养团没有被跨越实验室17标准化。此外，在蛋黄提供荧光脂质类似物可视运输和膳食脂类18的积聚，图像细胞成分包括既作为活体染料3和通过共价掺入复合脂脂滴，通过薄层色谱法调查代谢（TLC）19 </sup&gt;和高效液相色谱（HPLC）（SAF未发表数据），并提供总食物摄入量20的定量测定。

<table><tbody><tr><td>Tricaine (ethyl 3-aminobenzoate methanesulofnate salt)</td><td>Sigma-Aldrich</td><td>A5040-25G</td><td>Anesthesia for larval zebrafish</td></tr><tr><td>Chicken eggs</td><td>N/A</td><td>N/A</td><td>Organic, cage-free eggs, not enriched for omege-3 fatty acids</td></tr><tr><td>Ultrasonic processor 3000 sonicator</td><td>Misonix, Inc.</td><td>S-3000</td><td>To make egg yolk liposomes</td></tr><tr><td>Sonabox acoustic enclosure</td><td>Misonix, Inc.</td><td>432B</td><td>To make egg yolk liposomes</td></tr><tr><td>1/8&amp;rdquo; tapered microtip</td><td>Misonix, Inc.</td><td>419</td><td>To make egg yolk liposomes</td></tr><tr><td>Amber vials (4 ml, glass)</td><td>National Scientific</td><td>13-425</td><td>Lipid storage; includes vials, open-top caps, and cap septa</td></tr><tr><td>Incu-Shaker Mini&amp;nbsp;</td><td>Benchmark</td><td>1222U12</td><td>Incubated shaker for feeds</td></tr><tr><td>BODIPY FL C16&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>D3821</td><td>Fluorescent lipid analog; (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Hexadecanoic Acid)</td></tr><tr><td>BODIPY FL C12&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>D3822</td><td>Fluorescent lipid analog; (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Dodecanoic Acid)</td></tr><tr><td>BODIPY FL C5&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>D3834</td><td>Fluorescent lipid analog; (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Pentanoic Acid)</td></tr><tr><td>BODIPY FL C5</td><td>Thermo Fisher Scientific</td><td>D2183</td><td>Fluorescent lipid analog; (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Propionic Acid)</td></tr><tr><td>TopFluor cholesterol&amp;nbsp;</td><td>Avanti Polar Lipids Inc.</td><td>810255</td><td>Fluorescent lipid analog; 23-(dipyrrometheneboron difluoride)-24-norcholesterol</td></tr><tr><td>Fatty acid-free BSA</td><td>Sigma-Aldrich</td><td>A0281-1G</td><td>For TopFluor cholesterol solubilization</td></tr><tr><td>Methyl cellulose</td><td>Sigma-Aldrich</td><td>M0387</td><td>Mounting media for live larval imaging; 75 x 25 x 1 mm</td></tr><tr><td>Low melt agarose</td><td>Thermo Fisher Scientific</td><td>BP165-25</td><td>Mounting media for live larval imaging; 22 x 30</td></tr><tr><td>VWR microscope slides&amp;nbsp;</td><td>VWR&amp;nbsp;</td><td>16004-422</td><td>Mounting larvae for live imaging</td></tr><tr><td>Coverslips&amp;nbsp;</td><td>Cover Glass</td><td>12-544A</td><td>Mounting larvae for live imaging</td></tr><tr><td>Super glue</td><td>Loctite</td><td>LOC01-30379</td><td>Mounting larvae for live imaging</td></tr><tr><td>FluoroDish (glass bottom dish)</td><td>World Precision Instruments, Inc.&amp;nbsp;</td><td>FD35-100</td><td>Mounting larvae for live imaging; 35 mm dish, 23 mm glass, 0.17 mm glass thickness&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Confocal microscope</td><td>Leica Microsytems</td><td>SP-2, SP-5</td><td>Microscope for high magnification live imaging</td></tr><tr><td>Stereoscope</td><td>Nikon</td><td>SM21500</td><td>Microscope for low magnification live imaging</td></tr><tr><td>Glass culture tubes&amp;nbsp;</td><td>Kimble</td><td>73500-13100</td><td>Lipid extraction; (13 x 100 mm; 13 ml)</td></tr><tr><td>Savant SpeedVac Plus&amp;nbsp;</td><td>ThermoQuest</td><td>SC210A</td><td>Lipid extraction</td></tr><tr><td>Channeled TLC plates</td><td>Whatman Scientific</td><td>WC4855-821</td><td>Food intake assay; LK5D Silica Gel 150 A, 20 x 20 cm, 250 um thick; Discontinued</td></tr><tr><td>Channeled TLC plates</td><td>Analtech, Inc.</td><td>66911</td><td>Food intake assay; Direct replacement for Whatman Scientific TLC plates</td></tr><tr><td>Typhoon 9410 Variable Mode Imager</td><td>GE Healthcare</td><td>9410</td><td>Fluorescent plate reader for food intake assay</td></tr><tr><td>ImageQuant software</td><td>GE Healthcare</td><td>29000605</td><td>Analysis of food intake assay</td></tr><tr><td>5 3/4&amp;rsquo; Wide bore, borosilicate disposable pasteur pipets&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Kimble</td><td>63A53WT</td><td>Transfering larvae</td></tr></tbody></table>

high-fat feeding paradigm for larval zebrafish: feeding, live imaging, and quantification of food intake

Zebrafish are emerging as a model of dietary lipid processing and metabolic disease. This protocol describes how to feed larval zebrafish a lipid-rich meal, which consists of an emulsion of chicken egg yolk liposomes created by sonicating egg yolk in embryo media. Detailed instructions are provided to screen larvae for egg yolk consumption so that larvae that fail to feed will not confound experimental results. The chicken egg yolk liposomes can be spiked with fluorescent lipid analogs, including fatty acids and cholesterol, enabling both systemic and subcellular visualization of dietary lipid processing. Several methods are described to mount larvae that are conducive to short- and long-term live imaging with both upright and inverted objectives at high and low magnification. Additionally presented is an assay to quantify larval food intake by extracting the lipids of larvae fed fluorescent lipid analogs, spotting the lipids on a thin layer chromatography plate, and quantifying the fluorescence. Finally, critical aspects of the procedures, important controls, options for modifying the protocols to address specific experimental questions, and potential limitations are discussed. These techniques can be applied not only to focused, hypothesis driven inquiries, but also to a variety of screens and live imaging techniques to study dietary lipid metabolism and the control of food intake.

当在29-31℃的摇杆喂，大部分健康的幼虫（≥95％），将1小时内食用。一旦消耗蛋黄乳液，幼虫肠道变暗的颜色。非常暗的肠可以在2小时（ 图1）中观察到。如果幼虫喂食物或不喂，肠道是明确的。幼虫喂养蛋清表现出扩张肠腔中不颜色变暗。 <img alt="图1" src="/files/ftp_upload/54735/54735fig1.jpg" /> 图1:筛选幼虫食物摄入野生型6?...

Watch this Scientific Journal Video about High-fat Feeding Paradigm for Larval Zebrafish: Feeding, Live Imaging, and Quantification of Food Intake at JoVE.com

High-fat Feeding Paradigm for Larval Zebrafish: Feeding, Live Imaging, and Quantification of Food Intake

Zebrafish are emerging as a valuable model of dietary lipid processing and metabolic disease. Described are protocols of lipid-rich larval feeds, live imaging of dietary fluorescent lipid analogs, and quantification of food intake. These techniques can be applied to a variety of screening, imaging, and hypothesis driven inquiry techniques.

高脂饮食范式斑马鱼幼体:饲养，实时成像，和食物摄入的量化

Zebrafish are emerging as a model of dietary lipid processing and metabolic disease. This protocol describes how to feed larval zebrafish a lipid-rich ...

high-fat-feeding-paradigm-for-larval-zebrafish-feeding-live-imaging

Research

JoVE Journal

Biology

10.4K 次观看.  Carnegie Institution for Science. Zebrafish are emerging as a valuable model of dietary lipid processing and metabolic disease. Described are protocols of lipid-rich larval feeds, live imaging of dietary fluorescent lipid analogs, and quantification of food intake. These techniques can be applied to a variety of screening, imaging, and hypothesis driven inquiry techniques. 

视频: High-fat Feeding Paradigm for Larval Zebrafish: Feeding, Live Imaging, and Quantification of Food Intake

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and toxicological studies. Using automated digital tracking platforms, recent efforts in neuropharmacology are leveraging larval fish locomotor behaviors to identify potential therapeutic targets for novel small molecules. Similar to these efforts, research in the environmental sciences and comparative pharmacology and toxicology is examining various behaviors of fish models as diagnostic tools in tiered evaluation of contaminants and real-time monitoring of surface waters for contaminant threats. Whereas the zebrafish is a popular larval fish model in the biomedical sciences, the fathead minnow is a common larval fish model in ecotoxicology. Unfortunately, fathead minnow larvae have received considerably less attention in behavioral studies. Here, we develop and demonstrate a behavioral profile protocol using caffeine as a model neurostimulant. Though photomotor responses of fathead minnows were occasionally affected by caffeine, zebrafish&#160;were markedly more sensitive for photomotor and locomotor endpoints, which responded at environmentally relevant levels. Future studies are needed to understand comparative behavioral sensitivity differences among fish with age and time of day, and to determine whether similar behavioral effects would occur in nature and be indicative of adverse outcomes at the individual or population levels of biological organization.

Here, we present a protocol to examine larval zebrafish and fathead minnow locomotor activities and photomotor responses (PMR) using an automated tracking software. When incorporated in common toxicology bioassays, analyses of these behaviors provide a diagnostic tool to examine chemical bioactivity. This protocol is described using caffeine, a model neurostimulant.

检测幼虫行为反应剖面的实验协议: 在神经刺激咖啡因中的应用

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...

科研

环境科学

Analysis of Liver Microenvironment During Early Progression of Non-Alcoholic Fatty Liver Disease-Associated Hepatocellular Carcinoma in Zebrafish

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all liver cancer cases. With the introduction of effective treatments to prevent and treat hepatitis B/C, non-alcoholic fatty liver disease (NAFLD), and the more aggressive form know as non-alcoholic steatohepatitis (NASH), are quickly becoming the number one risk factors to develop HCC in modern societies. To better understand the role NASH has on the development of HCC we designed a NASH-associated HCC zebrafish. The optical clarity and genetic tractability of the zebrafish larvae make them an appealing and powerful model to study the liver microenvironment and immune cell composition using non-invasive fluorescent live imaging. This protocol describes how to use a NASH-associated HCC zebrafish model to investigate the effect of cholesterol surplus in the liver microenvironment and its impact on immune cell composition at early stages of the disease. First, we feed HCC larvae (s704Tg), which express hepatocyte-specific activated beta-catenin, with a 10% high cholesterol diet for 8 days to develop a NASH-associated HCC model. Here we describe how to make use of different transgenic lines to evaluate several early malignancy features in the liver by non-invasive confocal microscopy, such as liver area, cell, and nuclear morphology (hepatocytes area, nuclear area, nuclear:cytoplasmic ratio (N:C ratio), nuclear circularity, micronuclei/nuclear herniation scoring) and angiogenesis. Then, using transgenic lines with tagged immune cells (neutrophils, macrophages, and T cells) we show how to analyze liver immune cell composition in NASH-associated HCC larvae. The described techniques are useful to evaluate liver microenvironment and immune cell composition at early hepatocarcinogenesis stages, but they can also be modified to study such features in other liver disease models.

Here, we present how to generate a non-alcoholic fatty liver disease (NAFLD)-associated Hepatocellular Carcinoma (HCC) zebrafish model to study the impact of cholesterol surplus on liver microenvironment and immune cell landscape.

斑马鱼非酒精性脂肪肝相关肝细胞癌早期进展期间的肝脏微环境分析

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all ...

癌症研究

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system measures spontaneous behaviors and the response to an aversive stimulus, which is shown to the larvae via a PowerPoint presentation. The recorded images are analyzed with an ImageJ macro, which automatically splits the color channels, subtracts the background, and applies a threshold to identify individual larvae placement in the lanes. We can then import the coordinates into an Excel sheet to quantify swim speed, preference for edge or side of the lane, resting behavior, thigmotaxis, distance between larvae, and avoidance behavior. Subtle changes in behavior are easily detected using our system, making it useful for behavioral analyses after exposure to environmental toxicants or pharmaceuticals.

Our laboratory developed a novel high-throughput automated imaging system that is useful for the detection of several different behaviors in 7-day-old zebrafish larvae. The system can be used for detecting subtle changes in behavior after the larvae have been exposed to environmental toxicants or pharmaceuticals.

在斑马鱼幼虫的高通量自动化的行为分析

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system ...

神经科学

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval liver size in zebrafish HCC models provides a means to rapidly assess the effects of drugs and other manipulations on an oncogene-related phenotype. Here we show how to fix zebrafish larvae, dissect the tissues surrounding the liver, photograph livers using bright-field microscopy, measure liver area, and analyze results. This protocol enables rapid, precise quantification of liver size. As this method involves measuring liver area, it may underestimate differences in liver volume, and complementary methodologies are required to differentiate between changes in cell size and changes in cell number. The dissection technique described herein is an excellent tool to visualize the liver, gut, and pancreas in their natural positions for myriad downstream applications including immunofluorescence staining and in situ hybridization. The described strategy for quantifying larval liver size is applicable to many aspects of liver development and regeneration.

Here we demonstrate a method for quantifying liver size in larval zebrafish, providing a way to assess the effects of genetic and pharmacologic manipulations on liver growth and development.

使用光明场显微镜定量拉瓦尔斑马鱼的肝脏大小

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval ...

发育生物学

Real Time and Repeated Measurement of Skeletal Muscle Growth in Individual Live Zebrafish Subjected to Altered Electrical Activity

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish with fluorescently-marked plasma membranes can be scanned in a confocal laser scanning microscope in order to determine the volume of muscle tissue and the number of muscle fibers present. Efficient approaches for the measurement of cell number and size in live animals over time are described and validated against more arduous segmentation methods. Methods are described that permit the control of muscle electrical, and thus contractile, activity. Loss of skeletal muscle contractile activity greatly reduced muscle growth. In larvae, a protocol is described that allows reintroduction of patterned electrical-evoked contractile activity. The described methods minimize the effect of inter-individual variability and will permit analysis of the effect of electrical, genetic, drug, or environmental stimuli on a variety of cellular and physiological growth parameters in the context of the living organism. Long-term follow-up of the measured effects of a defined early-life intervention on individuals can subsequently be performed.

Optical clarity is a major advantage for cell biological and physiological work in zebrafish. Robust methods for measurement of cell growth in individual animals are described that permit novel insights into how growth of skeletal muscle and neighboring tissues are integrated with whole body growth.

实时和重复测量电活动改变的个体活斑马鱼的骨骼肌生长

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish ...

Using the Protozoan Paramecium caudatum as a Vehicle for Food-borne Infections in Zebrafish Larvae

Due to their transparency, genetic tractability, and ease of maintenance, zebrafish (Danio rerio) have become a widely-used vertebrate model for infectious diseases. Larval zebrafish naturally prey on the unicellular protozoan Paramecium caudatum. This protocol describes the use of P. caudatum as a vehicle for food-borne infection in larval zebrafish. P. caudatum internalize a wide range of bacteria and bacterial cells remain viable for several hours. Zebrafish then prey on P. caudatum, the bacterial load is released in the foregut upon digestion of the paramecium&#160;vehicle, and the bacteria colonize the intestinal tract. The protocol includes a detailed description of paramecia maintenance, loading with bacteria, determination of bacterial degradation and dose, as well as infection of zebrafish by feeding with paramecia. The advantage of using this method of food-borne infection is that it closely mimics the mode of infection observed in human disease, leads to more robust colonization compared to immersion protocols, and allows the study of a wide range of pathogens. Food-borne infection in the zebrafish model can be used to investigate bacterial gene expression within the host, host-pathogen interactions, and hallmarks of pathogenicity including bacterial burden, localization, dissemination and morbidity.

Using the Protozoan Paramecium caudatum as a Vehicle for Food-borne Infections in Zebrafish Larvae

Zebrafish (Danio rerio) are becoming a widely-used vertebrate animal model for microbial colonization and pathogenesis. This protocol describes the use of the protozoan Paramecium caudatum as a vehicle for food-borne infection in zebrafish larvae. P. caudatum readily internalizes bacteria and get taken up by larval zebrafish through natural preying behavior.

用原生动物给鱼作动物感染的载体

Due to their transparency, genetic tractability, and ease of maintenance, zebrafish (Danio rerio) have become a widely-used vertebrate model for ...

免疫与感染

Microgavage of Zebrafish Larvae

The zebrafish has emerged as a powerful model organism for studying intestinal development1-5, physiology6-11, disease12-16, and host-microbe interactions17-25. Experimental approaches for studying intestinal biology often require the in vivo introduction of selected materials into the lumen of the intestine. In the larval zebrafish model, this is typically accomplished by immersing fish in a solution of the selected material, or by injection through the abdominal wall. Using the immersion method, it is difficult to accurately monitor or control the route or timing of material delivery to the intestine. For this reason, immersion exposure can cause unintended toxicity and other effects on extraintestinal tissues, limiting the potential range of material amounts that can be delivered into the intestine. Also, the amount of material ingested during immersion exposure can vary significantly between individual larvae26. Although these problems are not encountered during direct injection through the abdominal wall, proper injection is difficult and causes tissue damage which could influence experimental results. We introduce a method for microgavage of zebrafish larvae. The goal of this method is to provide a safe, effective, and consistent way to deliver material directly to the lumen of the anterior intestine in larval zebrafish with controlled timing. Microgavage utilizes standard embryo microinjection and stereomicroscopy equipment common to most laboratories that perform zebrafish research. Once fish are properly positioned in methylcellulose, gavage can be performed quickly at a rate of approximately 7-10 fish/ min, and post-gavage survival approaches 100% depending on the gavaged material. We also show that microgavage can permit loading of the intestinal lumen with high concentrations of materials that are lethal to fish when exposed by immersion. To demonstrate the utility of this method, we present a fluorescent dextran microgavage assay that can be used to quantify transit from the intestinal lumen to extraintestinal spaces. This test can be used to verify proper execution of the microgavage procedure, and also provides a novel zebrafish assay to examine intestinal epithelial barrier integrity under different experimental conditions (e.g. genetic manipulation, drug treatment, or exposure to environmental factors). Furthermore, we show how gavage can be used to evaluate intestinal motility by gavaging fluorescent microspheres and monitoring their subsequent transit. Microgavage can be applied to deliver diverse materials such as live microorganisms, secreted microbial factors/toxins, pharmacological agents, and physiological probes. With these capabilities, the larval zebrafish microgavage method has the potential to enhance a broad range of research fields using the zebrafish model system.

We present a novel method for microgavage of larval zebrafish utilizing standard embryo microinjection and stereomicroscopy equipment. We demonstrate that microgavage is a safe and efficient technique useful for delivering controlled amounts of diverse materials specifically into the larval zebrafish intestinal lumen.

Microgavage斑马鱼幼虫

The zebrafish has emerged as a powerful model organism for studying intestinal development1-5, physiology6-11, disease12-16, and host-microbe ...

生物学

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

高通量<em&gt;斑马鱼</em&gt;能量消耗分析

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...

体内研究 斑马鱼幼虫的全脑成像

In Vivo Whole-Brain Imaging of Zebrafish Larvae Using Three-Dimensional Fluorescence Microscopy

As a vertebrate model animal, larval zebrafish are widely used in neuroscience and provide a unique opportunity to monitor whole-brain activity at the cellular resolution. Here, we provide an optimized protocol for performing whole-brain imaging of larval zebrafish using three-dimensional fluorescence microscopy, including sample preparation and immobilization, sample embedding, image acquisition, and visualization after imaging. The current protocol enables in vivo imaging of the structure and neuronal activity of a larval zebrafish brain at a cellular resolution for over 1 h using confocal microscopy and custom-designed fluorescence microscopy. The critical steps in the protocol are also discussed, including sample mounting and positioning, preventing bubble formation and dust in the agarose gel, and avoiding motion in images caused by incomplete solidification of the agarose gel and paralyzation of the fish. The protocol has been validated and confirmed in multiple settings. This protocol can be easily adapted for imaging other organs of a larval zebrafish.

作者聚焦：使用三维荧光显微镜对斑马鱼幼虫进行体内全脑成像  

Presented here is a protocol for in vivo whole-brain imaging of larval zebrafish using three-dimensional fluorescence microscopy. The experimental procedure includes sample preparation, image acquisition, and visualization.

体内 使用三维荧光显微镜对斑马鱼幼虫进行全脑成像

As a vertebrate model animal, larval zebrafish are widely used in neuroscience and provide a unique opportunity to monitor whole-brain activity at the ...

高脂饮食范式斑马鱼幼体:饲养，实时成像，和食物摄入的量化

摘要

探索更多视频

此视频中的章节

检测幼虫行为反应剖面的实验协议: 在神经刺激咖啡因中的应用

斑马鱼非酒精性脂肪肝相关肝细胞癌早期进展期间的肝脏微环境分析

在斑马鱼幼虫的高通量自动化的行为分析

使用光明场显微镜定量拉瓦尔斑马鱼的肝脏大小

实时和重复测量电活动改变的个体活斑马鱼的骨骼肌生长

用原生动物给鱼作动物感染的载体

Microgavage斑马鱼幼虫

高通量

体内使用三维荧光显微镜对斑马鱼幼虫进行全脑成像

体内使用三维荧光显微镜对斑马鱼幼虫进行全脑成像

高脂饮食范式斑马鱼幼体:饲养，实时成像，和食物摄入的量化

摘要

探索更多视频

此视频中的章节

检测幼虫行为反应剖面的实验协议: 在神经刺激咖啡因中的应用

斑马鱼非酒精性脂肪肝相关肝细胞癌早期进展期间的肝脏微环境分析

在斑马鱼幼虫的高通量自动化的行为分析

使用光明场显微镜定量拉瓦尔斑马鱼的肝脏大小

实时和重复测量电活动改变的个体活斑马鱼的骨骼肌生长

用原生动物给鱼作动物感染的载体

Microgavage斑马鱼幼虫

高通量

体内 使用三维荧光显微镜对斑马鱼幼虫进行全脑成像

体内 使用三维荧光显微镜对斑马鱼幼虫进行全脑成像

体内使用三维荧光显微镜对斑马鱼幼虫进行全脑成像

体内使用三维荧光显微镜对斑马鱼幼虫进行全脑成像