This work was supported by JSPS KAKENHI Grant Number 25860294 and 25590073. We would like to thank Ms. Yui Namie for the hand-drawn illustration, and Mr. Koshi Kataoka and Ms. Sayuri Ichikawa for assistance with the zebrafish maintenance.

所有动物的程序是由三重大学伦理委员会批准，并与国际准则根据日本动物福利法规“法对福利和动物管理”（日本国环境）进行，并遵守。 1.准备了针注：在麻醉状态下进行所有的实验，并尽一切努力，尽量减少痛苦。安乐死，鱼浸入冰 - 水浴（5份冰/ 1份水在≤4℃）≥20分钟。 <ol><li>通过抽1.0毫米外直径的玻璃毛细管用针拔出器（图1A）制备玻璃微毛细管针。 </li><li>切斜用细剪刀（ 图1B）针的提示。提供了理想尖端直径应该大约100 - 200微米（图1C）。如果尖地径r是太窄，血液不会进入针。 </li><li>溶解肝素生理盐水至5毫克/毫升的浓度。 </li><li>放置一个预切针在吸气管组件的鼻甲端和保持吸嘴在口中，或将针连接到灯泡分配器。浸入针尖到肝素溶液，并通过抽吸并通过溶液（图1D和1E）吹heparinize针头。 注意：吸气管组件已经用于斑马鱼精子冷冻保存16。长的橡胶管可以阻止任何血液飞入嘴。设置一个过滤器在所述管的中途可避免危险。 </li><li>存储在一个10厘米的培养皿中，然后风干的肝素化的针至少1小时。甲大量的针可以提前准备。 </li></ol> 2.麻醉<ol><li>通过将200个ml的鱼水与100微升2-苯氧乙醇制备的塑料小情况下，麻醉剂溶液（2-PE）。麻醉剂的终浓度为500ppm。 注意！ 2-PE具有快速的效果。 </li><li>除去鱼（AB株）从循环系统期望数量。 </li><li>用网鱼转移到麻醉剂1 - 2分钟（ 图1F）。观察鱼慢慢游，水平传播的胸鳍，一声惊呼，具有快速的鳃盖运动在1分钟内。 </li><li>随着时间的推移，观察鱼躺在外壳的底部，并最终停止游泳。当鱼停下来喘气和鳃盖动作缓慢麻醉的手术平面达到。在这一点上，鱼准备采血。 </li><li>使用撇渣器从2-PE抬起麻醉鱼并轻轻将其放置在纸巾浸渍于该麻醉药（图1G）。盖鱼的头部软组织文中还浸泡2-PE的解决方案，以防止眼睛干涩和使用另一种柔软的干纸巾轻轻擦干体表。 </li></ol> 3.采血<ol><li>发生在吸气管组件（或灯泡分配器）的管口端的肝素针和保持吸气管组件在嘴里的喉舌结束。 </li><li>把握鼻甲端和针一起，并小心地用针尖移除干扰鳞。将针头在30 - 45°角插入采血部位。避免胃肠道（ 图1H）穿刺。在使用灯泡分配器的，按用拇指和中指的灯泡，并阻断孔在与第一手指的灯泡的端部，再插入针如上​​所述。 注意：站点采血是沿着主体轴线和后向中的背主动脉的区域中的肛门。背主动脉（DA）和所述后部主静脉（PCV）只是腹侧脊柱（ 图2）。 </li><li>开始吸衔嘴吸引管组件的端部时的针毡接触脊柱。如果血液不涨，用手移动针尖巧妙地鼓励血流。需要注意的是，一旦血液上升到针，立即停止摇动吸轻轻地（ 图1I）。在使用灯泡分配器的，释放灯泡的压力以吸入血液。 </li><li>观察血液会慢慢上升到针以脉动方式不经抽吸，这是动脉血压，因为可能的。因此，就没有必要吸如果针正确地穿透动脉。 </li><li>停止抽吸采集的血液相应的卷后。从鱼取出针，用柔软的纸巾停止任何出血按下穿刺部位。 20秒的指压（图1J） -在大约10观察出血停止。 </li><li>后出血停止后，立即将鱼放回转移到CLE在温水（〜28℃）坦克。帮助鱼儿轻轻纷飞水对鱼鳃，直到它开始游泳恢复。 注意：保持高达5 postsampling鱼在2升罐和罐连接到循环系统充氧的鱼。除抗生素鱼水是没有必要的。保持正常的住房和喂鱼。 </li><li>排出血液从针放在一张封口膜（ 图1K）的洁净区。 </li><li>使用任何商业手持式血糖仪（ 图1L）测量血糖。 注意：血糖仪使用葡萄糖脱氢酶黄素腺嘌呤二核苷酸电极，并且需要的0.6微升的样品体积。 <ol><li>插入试纸完全进入计和直接接触血滴。自动观察血液抽取到测试条，将获得的血糖结果5秒上的显示区域。记录结果并丢弃测试条。用一个新的测试条为每个测量。 </li></ol></li><li> （可选步骤）采取准确量的血液由吸液和血液转移到微离心管，用于进一步的分析（血红蛋白，甘油三酯（TG），总胆固醇等 ）。如果需要的话，稀释的全血从一个钓鱼用盐水。离心血液样品3分钟，在680×g离心在RT和收获血浆。转移等离子体进新管中。在这一点上，它准备在生化分析中使用。 </li></ol>

<ol>
	<li>Lieschke, G., Currie, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+human+disease:+zebrafish+swim+into+view.">Animal models of human disease: zebrafish swim into view.</a> Nat Rev Genet. 8 (5), 353-367 (2007).</li><li>Penberthy, W. T., Shafizadeh, E., Lin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+as+a+model+for+human+disease.">The zebrafish as a model for human disease.</a> Front Biosci. 7, d1439-d1453 (2002).</li><li>Stachura, D. L., Traver, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+dissection+of+zebrafish+hematopoiesis.">Cellular dissection of zebrafish hematopoiesis.</a> Methods Cell Biol. 101, 75-110 (2011).</li><li>Jagadeeswaran, P., Sheehan, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+blood+coagulation+in+the+zebrafish.">Analysis of blood coagulation in the zebrafish.</a> Blood Cells Mol Dis. 25 (3-4), 239-249 (1999).</li><li>Jagadeeswaran, P., Sheehan, J. P., Craig, F. E., Troyer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+zebrafish+thrombocytes.">Identification and characterization of zebrafish thrombocytes.</a> Br J Haematol. 107 (4), 731-738 (1999).</li><li>Lieschke, G. J., Oates, A. C., Crowhurst, M. O., Ward, A. C., Layton, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphologic+and+functional+characterization+of+granulocytes+and+macrophages+in+embryonic+and+adult.">Morphologic and functional characterization of granulocytes and macrophages in embryonic and adult.</a> Blood. 98 (10), 3087-3096 (2001).</li><li>Iwanami, N. <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+understanding+the+evolution+of+the+vertebrate+immune+system+and+human+primary+immunodeficiency.">Zebrafish as a model for understanding the evolution of the vertebrate immune system and human primary immunodeficiency.</a> Exp Hematol. 42 (8), 697-706 (2014).</li><li>Babin, P. J., Goizet, C., Raldua, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+models+of+human+motor+neuron+diseases:+advantages+and+limitations.">Zebrafish models of human motor neuron diseases: advantages and limitations.</a> Prog Neurobiol. 118, 36-58 (2014).</li><li>Seth, A., Stemple, D. L., Barroso, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emerging+use+of+zebrafish+to+model+metabolic+disease.">The emerging use of zebrafish to model metabolic disease.</a> Dis Mod Mech. 6 (5), 1080-1088 (2013).</li><li>Eames, S. C., Philipson, L. H., Prince, V. E., Kinkel, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+sugar+measurement+in+zebrafish+reveals+dynamics+of+glucose+homeostasis.">Blood sugar measurement in zebrafish reveals dynamics of glucose homeostasis.</a> Zebrafish. 7 (2), 205-213 (2010).</li><li>Moss, J. B., 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=Regeneration+of+the+pancreas+in+adult+zebrafish.">Regeneration of the pancreas in adult zebrafish.</a> Diabetes. 58 (8), 1844-1851 (2009).</li><li>Oka, T., 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=Diet-induced+obesity+in+zebrafish+shares+common+pathophysiological+pathways+with+mammalian+obesity.">Diet-induced obesity in zebrafish shares common pathophysiological pathways with mammalian obesity.</a> BMC Physiol. 10 (21), (2010).</li><li>Chu, C. Y., 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=Overexpression+of+Akt1+enhances+adipogenesis+and+leads+to+lipoma+formation+in+zebrafish.">Overexpression of Akt1 enhances adipogenesis and leads to lipoma formation in zebrafish.</a> PLoS One. 7 (5), e36474 (2012).</li><li>Velasco-Santamar&#237;a, Y. M., Korsgaard, B., Madsen, S. S., Bjerregaard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bezafibrate,+a+lipid-lowering+pharmaceutical,+as+a+potential+endocrine+disruptor+in+male+zebrafish+(Danio+rerio).">Bezafibrate, a lipid-lowering pharmaceutical, as a potential endocrine disruptor in male zebrafish (Danio rerio).</a> Aquat Toxicol. 105 (1-2), 107-118 (2011).</li><li>Zang, L., Shimada, Y., Nishimura, Y., Tanaka, T., Nishimura, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel,+reliable+method+for+repeated+blood+collection+from+aquarium+fish.">A novel, reliable method for repeated blood collection from aquarium fish.</a> Zebrafish. 10 (3), 425-432 (2013).</li><li>Carmichael, C., Westerfield, M., Varga, Z. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+and+in+vitro+fertilization+at+the+zebrafish+international+resource+center.">Cryopreservation and in vitro fertilization at the zebrafish international resource center.</a> Methods Mol Biol. 546, 45-65 (2009).</li><li>Thorson, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+partitioning+of+body+water+in+Osteichthyes:+phylogenetic+and+ecological+implications+in+aquatic+vertebrates.">The partitioning of body water in Osteichthyes: phylogenetic and ecological implications in aquatic vertebrates.</a> Biol Bull-US. 120, 238-254 (1961).</li><li>Conte, F. P., Wagner, H. H., Harris, T. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+blood+volume+in+the+fish+(Salmo+gairdneri+gairdneri).">Measurement of blood volume in the fish (Salmo gairdneri gairdneri).</a> Am J Physiol. 205, 533-540 (1963).</li><li>Diehl, K. H., 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=A+good+practice+guide+to+the+administration+of+substances+and+removal+of+blood,+including+routes+and+volumes.">A good practice guide to the administration of substances and removal of blood, including routes and volumes.</a> J Appl Toxicol. 21 (1), 15-23 (2001).</li><li>Nahas, K., Provost, J. -. P., Baneux, P. H., Rabemampianina, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+acute+blood+removal+via+the+sublingual+vein+on+haematological+and+clinical+parameters+in+Sprague-Dawley+rats.">Effects of acute blood removal via the sublingual vein on haematological and clinical parameters in Sprague-Dawley rats.</a> Lab Anim. 34 (4), 362-371 (2000).</li><li>Curado, S., 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=Conditional+targeted+cell+ablation+in+zebrafish:+a+new+tool+for+regeneration+studies.">Conditional targeted cell ablation in zebrafish: a new tool for regeneration studies.</a> DevDyn. 236 (4), 1025-1035 (2007).</li><li>Andersson, O., 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=Adenosine+signaling+promotes+regeneration+of+pancreatic+beta+cells+in+vivo.">Adenosine signaling promotes regeneration of pancreatic beta cells in vivo.</a> Cell Metab. 15 (6), 885-894 (2012).</li><li>Hiramitsu, 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=Eriocitrin+ameliorates+diet-induced+hepatic+steatosis+with+activation+of+mitochondrial+biogenesis.">Eriocitrin ameliorates diet-induced hepatic steatosis with activation of mitochondrial biogenesis.</a> Sci Rep-UK. 4, 3708 (2014).</li><li>Zang, L., Shimada, Y., Kawajiri, J., Tanaka, T., Nishimura, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Yuzu+(Citrus+junos+Siebold+ex+Tanaka)+peel+on+the+diet-induced+obesity+in+a+zebrafish+model.">Effects of Yuzu (Citrus junos Siebold ex Tanaka) peel on the diet-induced obesity in a zebrafish model.</a> J Funct Foods. 10, 499-510 (2014).</li><li>Schlegel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+non-alcoholic+fatty+liver+disease+with+zebrafish:+a+confluence+of+optics,+genetics,+and+physiology.">Studying non-alcoholic fatty liver disease with zebrafish: a confluence of optics, genetics, and physiology.</a> Cell Mol Life Sci. 69 (23), 3953-3961 (2012).</li><li>Stoletov, K., 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=Vascular+lipid+accumulation,+lipoprotein+oxidation,+and+macrophage+lipid+uptake+in+hypercholesterolemic+zebrafish.">Vascular lipid accumulation, lipoprotein oxidation, and macrophage lipid uptake in hypercholesterolemic zebrafish.</a> Circ Res. 104 (8), 952-960 (2009).</li><li>Thomas, C. D., 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=Nutrient+balance+and+energy+expenditure+during+ad+libitum+feeding+of+high-fat+and+high-carbohydrate+diets+in+humans.">Nutrient balance and energy expenditure during ad libitum feeding of high-fat and high-carbohydrate diets in humans.</a> Am J Clin Nutr. 55 (5), 934-942 (1992).</li></ol>

The authors have nothing to disclose.

我们在座的详细协议，用于连续获得血液从成年斑马鱼。这种方法是简单的进行，我们使用它在实验室，每天。此采血方法是基于插入玻璃毛细管针进入斑马鱼的背主动脉。在此过程中，关键是要小心不烧蚀脊柱，因为它是标准用于搜索背主动脉。降低颈椎损伤会提高患者的生存率。虽然这种技术是简单，易于掌握，有可以保证高的成功率和存活率的最佳做法。一个熟练的研究员将需要1-2分钟进?...

斑马鱼正日益普及人类疾病的一种有价值的模式，因为他们的器官和遗传学类似于人类1,2。在发育生物学的领域中，许多研究已经证明，斑马鱼和人显示标记相似造血3，止血4,5，和髓6。成年斑马鱼也用于研究免疫学7，神经变性8和肥胖相关疾病9，因为此模式生物股与破坏在人类疾病共同通路。肥胖和肥胖相关疾病（糖尿病，脂肪肝和酒精性脂肪性肝炎和动脉粥样硬化），斑马鱼血糖和血脂水平已经在几个转基因和饮食诱导的肥胖模型10月13日被彻底调查。 从单个动物反复采血会减少动物的使用和DECrease个体差异。然而，重复的样品采集是因为它们具有相对小的血容量和缺乏方便船舶在小动物，如斑马鱼在技术上困难。已经开发了几种方法用于从斑马鱼一次性采血，虽然这些方法有其自身的缺陷，包括致命性，相关的组织损伤和有限的血容量。例如，1-5微升血液可以从约0.3厘米的横向切口，在背主动脉5的区域收获长度。斩首用剪刀通过肩带切割可收集5-10微升血液10。另一种方便的采血方法是尾切除14。心脏穿刺是用于从相同的鱼重复采血一个潜在的替代方法，但非常小的量，得到（约50 NL）与此过程限制的分析，可以是perfor的数量配有11。因此，新的协议需要启用反复的非致命的血液采样，这将是一个重要的进步所必需的这种微生物是一个标准的模式生物的人类疾病。这种技术将允许用于测试药理反应，发现分子生物标记物的诊断，确定预后，以及各种疾病，如代谢疾病，退行性疾病和多种类型的恶性肿瘤的监测。 因此，我们开发了获得血液从斑马鱼连续15微创方法。在这里，我们演示程序可视化，并提供该技术的详细协议。使用这种方法，基于各种参数，包括血红蛋白，空腹血糖，并在健康成年斑马鱼的血脂正常值进行了评价。此外，我们还评估了此方法是否适用于需要通过米系列样本的研究期间过度喂食实验onitoring中血糖水平的瞬时变化。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Glass capillaries with filament</td>
			<td>Narishige</td>
			<td>GD-1</td>
			<td>1.0-mm-outer-diameter.</td>
		</tr>
		<tr>
			<td>Needle puller</td>
			<td>Narishige</td>
			<td>PC-10</td>
			<td>To produce the needls</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Wako Pure Chemical Industries</td>
			<td>081-00136</td>
			<td>For heparinization</td>
		</tr>
		<tr>
			<td>Aspirator tube assembly</td>
			<td>Drummond</td>
			<td>2-040-000</td>
			<td>For blood collection</td>
		</tr>
		<tr>
			<td>Bulb dispenser</td>
			<td>Drummond</td>
			<td>1-000-9000</td>
			<td>For blood collection</td>
		</tr>
		<tr>
			<td>2-phenoxyethanol</td>
			<td>Wako Pure Chemical Industries</td>
			<td>163-12075</td>
			<td>For anesthetizing the fish</td>
		</tr>
		<tr>
			<td>DRI-CHEM3500V</td>
			<td>Fujifilm</td>
			<td>-</td>
			<td>For hemoglobin measurement</td>
		</tr>
		<tr>
			<td>DRI-CHEM Slides</td>
			<td>Fujifilm</td>
			<td>Hb-WII</td>
			<td>For hemoglobin measurement</td>
		</tr>
		<tr>
			<td>Glutest Neo Super</td>
			<td>Sanwa Kagaku Kenkyusho</td>
			<td>-</td>
			<td>For bood glucose measurement</td>
		</tr>
		<tr>
			<td>Wako L-type TG kit</td>
			<td>Wako Pure Chemical Industries</td>
			<td>464-44201</td>
			<td>For TG measurement</td>
		</tr>
		<tr>
			<td>Wako L-type CHO kit</td>
			<td>Wako Pure Chemical Industries</td>
			<td>460-44301</td>
			<td>For total cholesterol measurement</td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Alcan Packaging</td>
			<td>PM996</td>
			<td>To expel the blood on</td>
		</tr>
	</tbody>
</table>

repeated blood collection for blood tests in adult zebrafish

重复采血是实验室动物进行的最普遍的技术之一。但是，还没有建立一个非致命性协议的采血从斑马鱼。以前的方法采血从斑马鱼是致命的，如外侧切口，斩首和尾巴切除。因此，我们已经开发了一种新的“重复”的血液采集方法，以及在座的详细协议，概述了此过程。这种方法是微创并导致非常低死亡率（2.3％），用于斑马鱼，从而使来自同一个体的重复采血。血液取样的最大体积是依赖于体重的鱼。间隔重复采集血样的体积应每周体重或≤1％，每2周，这是由血液中的血红蛋白的测定评价≤0.4％。此外，血红蛋白，空腹血糖，血浆甘油三酯（TG）和总碳在男性和女性的成年斑马鱼holesterol水平进行了测量。我们也应用这个方法来研究糖代谢饮食诱导的肥胖的失调。这种血液采集方法将允许许多应用，包括葡萄糖和脂质代谢和血液学研究，这将增加使用斑马鱼作为人类疾病模型有机体。

该采血方法导致最少伤害的斑马鱼（A &lt;1毫米穿刺; 图1J），并产生一个非常低的死亡率为2.3％。我们研究了血液，可以从一个单一的鱼进行收集和评估的关系，它的体重（图3）的最大容积。我们发现，收集到的最大的血容量呈线性体重（R = 0.813）相关。的血液从个体鱼（体重=1.071克）收集到的最大体积为25微升，并最小体积是从鱼体重0.115克1.3微升。这表明，血液收集?...

Watch this Scientific Journal Video about Repeated Blood Collection for Blood Tests in Adult Zebrafish at JoVE.com

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

重复采血车在成年斑马鱼验血

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

repeated-blood-collection-for-blood-tests-in-adult-zebrafish

Research

JoVE Journal

Biology

34.5K 次观看.  Mie University. Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

视频: Repeated Blood Collection for Blood Tests in Adult Zebrafish

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

从美国的马蹄蟹，鲎波吕斐摩斯采血车

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

科研

生物学

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

从新生小鼠的肺血管内皮细胞的分离和培养

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

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

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

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

全安装胚胎肢体皮肤血管的免疫组织化学分析：一个模型系统，研究在胚胎血管分支形态发生

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

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 ...

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

在成年斑马鱼的生化分析采血车

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

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 ...

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

对于耐药性突变对激酶抑制剂的筛选和验证方法

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

对于外小泡的使用流式细胞仪分析技术

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.1-4 Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells.

Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol fixation).