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 border="0" cellpadding="0" cellspacing="0" width="832">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">Tricaine (ethyl 3-aminobenzoate methanesulofnate salt)</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:127px;">A5040-25G</td>
			<td style="width:335px;">Anesthesia for larval zebrafish</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Chicken eggs</td>
			<td style="width:143px;">N/A</td>
			<td style="width:127px;">N/A</td>
			<td>Organic, cage-free eggs, not enriched for omege-3 fatty acids</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Ultrasonic processor 3000 sonicator</td>
			<td style="width:143px;">Misonix, Inc.</td>
			<td style="width:127px;">S-3000</td>
			<td>To make egg yolk liposomes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Sonabox acoustic enclosure</td>
			<td style="width:143px;">Misonix, Inc.</td>
			<td style="width:127px;">432B</td>
			<td>To make egg yolk liposomes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">1/8&#8221; tapered microtip</td>
			<td style="width:143px;">Misonix, Inc.</td>
			<td style="width:127px;">419</td>
			<td>To make egg yolk liposomes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Amber vials (4 ml, glass)</td>
			<td style="width:143px;">National Scientific</td>
			<td style="width:127px;">13-425</td>
			<td>Lipid storage; includes vials, open-top caps, and cap septa</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Incu-Shaker Mini&#160;</td>
			<td style="width:143px;">Benchmark</td>
			<td style="width:127px;">1222U12</td>
			<td>Incubated shaker for feeds</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">BODIPY FL C16&#160;</td>
			<td style="width:143px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">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 height="40">
			<td height="40" style="height:40px;width:231px;">BODIPY FL C12&#160;</td>
			<td style="width:143px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">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 height="40">
			<td height="40" style="height:40px;width:231px;">BODIPY FL C5&#160;</td>
			<td style="width:143px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">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 height="40">
			<td height="40" style="height:40px;width:231px;">BODIPY FL C5</td>
			<td style="width:143px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">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 height="40">
			<td height="40" style="height:40px;width:231px;">TopFluor cholesterol&#160;</td>
			<td style="width:143px;">Avanti Polar Lipids Inc.</td>
			<td style="width:127px;">810255</td>
			<td>Fluorescent lipid analog; 23-(dipyrrometheneboron difluoride)-24-norcholesterol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Fatty acid-free BSA</td>
			<td>Sigma-Aldrich</td>
			<td style="width:127px;">A0281-1G</td>
			<td>For TopFluor cholesterol solubilization</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Methyl cellulose</td>
			<td>Sigma-Aldrich</td>
			<td style="width:127px;">M0387</td>
			<td>Mounting media for live larval imaging; 75 x 25 x 1 mm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">Low melt agarose</td>
			<td style="width:143px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">BP165-25</td>
			<td>Mounting media for live larval imaging; 22 x 30</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">VWR microscope slides&#160;</td>
			<td style="width:143px;">VWR&#160;</td>
			<td style="width:127px;">16004-422</td>
			<td>Mounting larvae for live imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Coverslips&#160;</td>
			<td style="width:143px;">Cover Glass</td>
			<td style="width:127px;">12-544A</td>
			<td>Mounting larvae for live imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Super glue</td>
			<td style="width:143px;">Loctite</td>
			<td style="width:127px;">LOC01-30379</td>
			<td>Mounting larvae for live imaging</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">FluoroDish (glass bottom dish)</td>
			<td style="width:143px;">World Precision Instruments, Inc.&#160;</td>
			<td style="width:127px;">FD35-100</td>
			<td>Mounting larvae for live imaging; 35 mm dish, 23 mm glass, 0.17 mm glass thickness&#160;&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Confocal microscope</td>
			<td>Leica Microsytems</td>
			<td style="width:127px;">SP-2, SP-5</td>
			<td>Microscope for high magnification live imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Stereoscope</td>
			<td style="width:143px;">Nikon</td>
			<td style="width:127px;">SM21500</td>
			<td>Microscope for low magnification live imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Glass culture tubes&#160;</td>
			<td style="width:143px;">Kimble</td>
			<td style="width:127px;">73500-13100</td>
			<td>Lipid extraction; (13 x 100 mm; 13 ml)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Savant SpeedVac Plus&#160;</td>
			<td style="width:143px;">ThermoQuest</td>
			<td style="width:127px;">SC210A</td>
			<td>Lipid extraction</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Channeled TLC plates</td>
			<td style="width:143px;">Whatman Scientific</td>
			<td style="width:127px;">WC4855-821</td>
			<td>Food intake assay; LK5D Silica Gel 150 A, 20 x 20 cm, 250 um thick; Discontinued</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Channeled TLC plates</td>
			<td style="width:143px;">Analtech, Inc.</td>
			<td style="width:127px;">66911</td>
			<td>Food intake assay; Direct replacement for Whatman Scientific TLC plates</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Typhoon 9410 Variable Mode Imager</td>
			<td style="width:143px;">GE Healthcare</td>
			<td style="width:127px;">9410</td>
			<td>Fluorescent plate reader for food intake assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">ImageQuant software</td>
			<td style="width:143px;">GE Healthcare</td>
			<td>29000605</td>
			<td>Analysis of food intake assay</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">5 3/4&#8217; Wide bore, borosilicate disposable pasteur pipets&#160;&#160;&#160;</td>
			<td style="width:143px;">Kimble</td>
			<td style="width:127px;">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

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

科研

生物学

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

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

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

现场共聚焦显微镜对斑马鱼的胚胎脑成像

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

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

移植绿色荧光蛋白表达对视网膜和大脑发育的实时成像嵌合斑马鱼胚胎的卵裂球

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function. Our studies have found that dying cells send signals to their live neighbors to form and contract a ring of actin and myosin that ejects it out from the epithelial sheet while closing any gaps that might have resulted from its exit, a process termed cell extrusion1. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe a method to induce and image extrusion in the larval zebrafish epidermis. To visualize extrusion, we inject a red fluorescent protein labeled probe for F-actin into one-cell stage transgenic zebrafish embryos expressing green fluorescent protein in the epidermis and induce apoptosis by addition of G418 to larvae. We then use time-lapse imaging on a spinning disc confocal microscope to observe actin dynamics and epithelial cell behaviors during the process of apoptotic cell extrusion. This approach allows us to investigate the extrusion process in live epithelia and will provide an avenue to study disease states caused by the failure to eliminate apoptotic cells.

Dying cells are extruded from epithelial tissues by concerted contraction of neighboring cells without disrupting barrier function. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe methods to induce and image extrusion in the larval zebrafish epidermis at cellular resolution.

发展中国家斑马鱼的表皮细胞挤压实时成像

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function.  Our studies have found ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

检测现场的<em&gt;大肠杆菌</em&gt; O157:H7细胞的PMA-qPCR的

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

测量水润谷胱甘肽诱导饲养响应

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution.

实时在体记录了拟南芥钙信号在使用荧光传感器的昆虫饲喂时

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant ...

A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes

The mosquito midgut harbors a highly dynamic microbiome that affects the host metabolism, reproduction, fitness, and vector competence. Studies have been conducted to investigate the effect of gut microbes as a whole; however, different microbes could exert distinct effects toward the host. This article provides the methodology to study the effect of each specific mosquito gut microbe and the potential mechanism.
This protocol contains two parts. The first part introduces how to dissect the mosquito midgut, isolate cultivable bacteria colonies, and identify bacteria species. The second part provides the procedure to generate antibiotic-treated mosquitoes and reintroduce one specific bacteria species.

This article presents a protocol to investigate the effect of individual mosquito gut bacteria, including isolation and identification of mosquito midgut cultivable microbes, antibiotic depletion of mosquito gut bacteria, and reintroduce one specific bacteria species.

抗生素处理蚊子的细菌口服喂养测定

The mosquito midgut harbors a highly dynamic microbiome that affects the host metabolism, reproduction, fitness, and vector competence. Studies have been ...

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

Malaria remains one of the most important public health problems, causing significant morbidity and mortality. Malaria is a mosquito borne disease transmitted through an infectious bite from the female Anopheles mosquito. Malaria control will eventually rely on a multitude of approaches, which includes ways to block transmission to, through and from mosquitoes. To study mosquito stages of malaria parasites in the laboratory, we have optimized a protocol to culture highly infectious Plasmodium falciparum gametocytes, a parasite stage required for transmission from the human host to the mosquito vector. P. falciparum gametocytes mature through five morphologically distinct steps, which takes approximately 1-2 weeks. Gametocyte culture described in this protocol is completed in 15 days and are infectious to mosquitoes from days 15-18. These protocols were developed to maintain a continuous cycle of infection competent gametocytes and to maintain uninterrupted supply of mosquito stages of the parasite. Here, we describe the methodology of gametocyte culture and how to infect mosquitoes with these parasites using glass membrane feeders.

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

Detailed investigations on mosquito stages of malaria parasites are critical to design effective transmission blocking strategies. This protocol demonstrates how to effectively culture infectious gametocytes and then feed these gametocytes to mosquitoes to generate mosquito stages of P. falciparum.

恶性疟原虫 通过人工膜喂养进行配子体培养和蚊子感染

Malaria remains one of the most important public health problems, causing significant morbidity and mortality. Malaria is a mosquito borne disease ...

Live Imaging of Microtubule Dynamics in Glioblastoma Cells Invading the Zebrafish Brain

With a dismal median survival time in real populations-between 6 to 15 months-glioblastoma (GBM) is the most devastating malignant brain tumor. Treatment failure is mainly due to the invasiveness of GBM cells, which speaks for the need for a better understanding of GBM motile properties. To investigate the molecular mechanism supporting GBM invasion, new physiological models enabling in-depth characterization of protein dynamics during invasion are required. These observations would pave the way to the discovery of novel targets to block tumor infiltration and improve patient outcomes. This paper reports how an orthotopic xenograft of GBM cells in the zebrafish brain permits subcellular intravital live imaging. Focusing on microtubules (MTs), we describe a procedure for MT labeling in GBM cells, microinjecting GBM cells in the transparent brain of 3 days post fertilization (dpf) zebrafish larvae, intravital imaging of MTs in the disseminating xenografts, altering MT dynamics to assess their role during GBM invasion, and analyzing the acquired data.

We report a technique permitting live imaging of microtubule dynamics in glioblastoma (GBM) cells invading a vertebrate brain tissue. Coupling the orthotopic injection of fluorescently tagged GBM cells into a transparent zebrafish brain with high-resolution intravital imaging allows the measurement of cytoskeleton dynamics during in situ cancer invasion.

侵入斑马鱼大脑的胶质母细胞瘤细胞微管动力学的实时成像

With a dismal median survival time in real populations-between 6 to 15 months-glioblastoma (GBM) is the most devastating malignant brain tumor. Treatment ...