We thank Julie R. Perlin for helpful comments on the manuscript. D.I.S. is supported by grants from the American Society of Hematology, the Cooley's Anemia Foundation, and the NIH (K01DK085217 and R03DK100672). E.J.H. is a Howard Hughes Medical Institute Fellow of the Helen Hay Whitney Foundation. B.L. is a Howard Hughes Medical Institute Medical Research Fellow. B.W.B. is supported by an Irvington Fellowship from the Cancer Research Institute and a Young Investigator Award from the Conquer Cancer Foundation of ASCO. L.I.Z. is supported by grants from the NIH (R01CA103846, P01HL032262, and R01HL04880), Taub Foundation for MDS Research, Harvard Stem Cell Institute, and is an investigator of the Howard Hughes Medical Institute.

该协议被批准波士顿儿童医院的动物护理和使用委员会。这个协议是从以 ​​前出版的方法15进行修改。 1.试剂的制备（提前几天或几周） <ol><li>准备1.5％琼脂糖涂层的菜肴。 添加1.5克琼脂糖至100ml斑马鱼的E3介质中的Erlenmeyer烧瓶中。热火，琼脂糖溶解，再倒入约5mL成直径100毫米×20毫米深的培养皿中。 注意:这些都是用于胚胎dechorionation（步骤3.1），并为联体手术（步骤3.3）。在4℃保存琼脂糖涂层菜的几个星期;带他们出去了冰箱和联体手术当天早晨温至室温。 </li><li>制备4％的甲基纤维素。 将4-在100ml的E3的甲基纤维素粉末在锥形瓶中搅拌棒。将在4℃的搅拌盘烧瓶中，设置为最低速度，让它搭配为2天。 <ol><li>间歇用刮刀分手甲基纤维素的白色团块。甲基纤维素的结晶不充分溶解在该浓度。当该溶液变得清晰并出现均匀，没有白色团块残留，等分试样在-20℃下长期贮存溶液进入1.5毫升离心管并冷冻。 注意:甲基纤维素的较低浓度都可以使用;然而，这种较高的百分比，更粘稠甲基纤维素已取得了最好的结果。 </li></ol></li><li>准备高钙林格（HCR）解决方案。 让400毫升HCR溶液，混合8毫升的5M NaCl（116毫最终），400微升3M氯化钾（2.9毫最终），800微升5M的CaCl 2（10mM的终浓度），以及1M HEPES的2毫升（5毫米最终出）与390毫升E3媒体。 注:在4℃下保存HCR解决了几个星期;温至室温连体手术当天的早上。 </li><li>准备5 - 6传输修改巴斯德移液器环囊胚对。简要地加热该吸管在本生灯的结束。然后，使用大钳，弯曲吸管至约45℃的端，而它仍然是热的。 <ol><li>为了确保有可能损坏胚胎，简要地握住吸管尖在火焰周边3秒没有锋利的边缘。这将平滑/抛光移液器的结束。此改性的玻璃巴斯德移液管一起使用，在下面的步骤3.5 10毫升吸管泵（ 图1）。 </li></ol></li><li>准备玻璃针工具囊胚对手术缝合。作为斑马鱼胚胎显微注射完成3针玻璃 - 拉2。使用实验室膜各玻璃针固定到木杆或塑料处理取笑针（ 图1A）的末尾。使用镊子，断绝了针的末端处直径约20 - 30微米。使用一个阶段微米到引导该步骤（图1B）。 注:网元的大小edle前端是重要的。如果前端是太大，难以精确地控制，并且可以引起过度的破坏。如果尖端太小则难以充分地缠绕在胚胎。 </li></ol> 2.胚胎斑马鱼和收集繁殖对设置（-1天0天） <ol><li>建立成年斑马鱼的繁殖对。设置鱼夜联体手术前要进行的。使用分隔分离男性和女性。为了增加期望的胚胎，将获得，设置若干对每一行的可能性。我们发现，一般为20 - 在对50％的产卵。 </li><li>第二天早上，拉分频器，允许交配。收集用细网滤茶任何胚胎，然后将它们转移到含有E3胚胎介质18培养皿。 </li><li>根据需要微注射胚胎（ 例如，用25 PG质粒DNA，200 PG的mRNA，或用1 - 6纳克啉）收集的1小时之内。让他们生长，直到256细胞阶段。如果DNA表达构建体放置在热休克启动子的控制下（ 例如，热休克蛋白70， 图4B），由热震惊parabionts在37℃下控制的基因表达的定时在后一阶段（ 例如 ，在36高倍视野）。 注:对于荧光转基因的替代，荧光葡聚糖可以加入到DNA，RNA或吗啉代喷射混合（ 例如，葡聚糖，级联蓝在2毫克/毫升）。葡聚糖染料不会正常发展干扰和将允许适当的荧光联体融合后下被识别的注入胚。可替代地，着色菌株的联体（ 例如，AB）与非着色的应变（ 例如，卡斯帕）可以用来在稍后阶段，以确定所注入的胚胎。 </li><li>孵育胚胎在28.5°C.Use一个立体镜定期监测其发展，因为他们256细胞附近开发elopmental阶段（约2.5 HPF）。 注意:如果需要，转移胚胎到不同的温度，以确保联体伙伴（ 例如，吗啉代注射的胚胎往往发展比其未注射的同行慢移的未注射的胚胎至RT之间阶段匹配而啉注射的胚胎放置。在28.5℃，将导致其阶段几个小时的发展）调整后。 </li></ol> 3.联体斑马鱼胚胎的产生通过发展囊胚的手术融合<ol><li>作为胚胎接近256细胞阶段（约2.5 HPF），从每一个遗传背景（ 即，遗传突变，转基因系和/或基因操作）转移胚胎成单独的琼脂糖包被的培养皿（ 例如，一个盘用于吗啉代注射胚胎和对照胚胎第二盘）。另外，胚胎转移到清洁，无划痕的玻璃烧杯或玻璃培养皿。 注意:避免使用无涂层的塑料菜肴胚胎会坚持到塑料和被他们的绒毛膜一旦损坏。 </li><li>倒出E3媒体直到刚好足够的液体仍然覆盖胚胎（约7 - 10ml）中。加入200微升50毫克/从灰色链霉菌 （蛋白酶）的细胞外液中分离蛋白酶毫升混合物中。摇动轻轻胚胎每隔几分钟。 <ol><li>当胚胎的50％已经走出自己的绒毛膜，这通常需要5 - 新鲜的E3胚胎中期的 - （20毫升约15）10分钟，丰盛的体积冲洗一下三次。 <ol><li>轻轻拉起来到改性玻璃巴斯德吸管，然后轻轻地消除他们Dechorionate留在他们的绒毛膜任何胚胎。一旦胚胎已dechorionated和清洗，更换HCR解决方案的E3媒体。作为替代蛋白酶，用细镊子手动删除绒毛膜。目标是在60 - 100 dechor可用于每个实验条件ionated胚胎。 注意:这是几倍大于最终将使用更多的，但许多胚胎有可能处理或手术联体期间被损坏。一旦胚dechorionated，保持它们浸没，并且不使它们接触水的表面上，作为表面张力将导致他们被破坏。请记住，使用高剂量的蛋白酶使胚胎走出自己的绒毛膜更快，更同步的时尚。当使用蛋白酶的较低剂量，将胚需要更长的时间从绒毛膜出现并在一个不太同步方式，其可导致胚胎的一部分通过长时间暴露于蛋白酶被损坏这样做。 </li></ol></li></ol></li><li>解冻在最大速度台式离心机的甲基纤维素（在上面的步骤1.2。制造），离心10分钟以沉淀不溶的结晶，这将损坏胚胎或破裂的蛋黄秒。 <ol><li>离心后，用明确的上限比例（体积约75％）。转让1 - 甲基纤维素的1.5厘米直径的滴成被覆琼脂糖1.5％培养皿。小心避免从管的底部起草任何丸状晶体。 </li><li>使用一个标记，以预先在盘的底侧每个液滴的位置。将12 - 每100毫米直径的培养皿甲基纤维素 - （3等分的价值2）15滴。准备根据需要每个实验尽可能多的板块。 注:使用每个液滴为单囊胚对的融合。该标记将指定每个液滴一旦介质被添加-在该点液滴变得几乎不可见的位置。 </li></ol></li><li>准备每个步骤3.3以上制备琼脂糖涂层菜抗生素的补充解决方案，HCR 1个40毫升一份。以各40毫升等分试样的HCR中的50单位/毫升，氨苄青霉素，50单位/毫升，和卡那霉素加青霉素 - 链霉素，在0.5微克/毫升。 <ol><li>当准备进行囊胚融合，认真填写每个含甲基纤维素滴用40毫升抗生素补充HCR解决方案的菜肴。如果HCR溶液中加入太快，它可以破坏液滴。 </li></ol></li><li>附加改性的玻璃巴斯德吸管（将在上面步骤1.4制），以10毫升吸管泵。在一个立体镜收集来自各个背景（ 例如，一个吗啉注射胚胎和一个野生型胚胎）一dechorionated胚胎。 <ol><li>分配的两个胚胎之前，使用巴斯德吸管，使在甲基纤维素降的中间小凹陷，然后轻轻既胚胎存入抑郁症。 注意:在传送过程中，转动巴斯德吸管略微向上，使胚胎沉入巴斯德移液管的弯曲，以便避免与在移液管的顶部或底部的液体的表面的制造接触（这可能会导致到兵卫- [R破裂）。 </li></ol></li><li>沉积胚胎成甲基纤维素后，立即用木杆或塑料处理取笑针上的端部（ 图1A）的凝胶加载吸管尖小心重新定位甲基纤维素中的胚胎，使得它们的动物极点是直接抚摸对方。 <ol><li>利用通过甲基纤维素接近胚胎斯文扫地的动作对其重新定位，而不直接接触他们。让胚胎坐5分钟，使甲基纤维素会沉淀在他们身边。在此期间，装载下一对成另一种降。 注意事项:避免让与胚胎直接接触，特别注意不要触摸卵黄囊，这将容易破裂的任何部分。 </li></ol></li><li>使用玻璃针的工具，小心翼翼地绕每个胚胎在其接触点。用温和的缝制动作中，从第一胚胎细胞可以再次被拉入第二胚胎，然后。在这个运动结束，举行的地方针2 - 3秒，然后绘制它拿走非常缓慢。 注:有一个周期约2 - 3秒后的胚受伤其中两种组织似乎更粘合剂，更可能连接到彼此。因此，我们认为，保持到位针伤人后简要地有助于保持每个胚的伤员组织中的时间的初期接触伤人之后。 </li><li>允许这些胚胎坐10 - 在室温下15分钟。在此期间，不断地缝合在甲基纤维素的其他滴新对。 10后- 15分钟，评估所述第一对是否保持附着（ 图2A）。 注意:如果胚胎更长附着，重复缝合过程只要胚胎大约30％的外包阶段比年轻。这通常允许最多三次尝试胚胎缝合在一起。如果胚胎似乎一直保持附件但Connection并不是实质性的，在以加强它的连接的边缘执行附加缝合。避免摇动或在连体手术和潜伏期移动菜肴。 注意:尽管他们的早期发育阶段和感知的脆弱性，胚胎细胞块可以容忍操纵的大量（ 即，伤人和缝合），仍然正常发育。蛋黄，但是，可以只用轻轻一碰破裂，杀死胚胎。当拼接两个囊胚的玻璃针，不要使每个胚胎的细胞质量和蛋黄之间的界面接触。 </li><li>在同样的菜和媒体（不改变媒体）孵育胚胎O / N为28.5℃。到了第二天早上，胚胎将有大部分溶解甲基纤维素液滴搬出去了。删除未成功融合的胚胎。倒出培养基，并用新鲜25毫升E3替换。 注意:如果使用工作着色应变，加入500μl0.15％（50倍）苯基硫脲（PTU）至终浓度0.003％到方框色素沉着。胚胎需要在PTU仍然持续保持色素沉着的抑制。 </li></ol> 4.显微镜装置，图像采集，处理及分析<ol><li>制备0.8％的低熔点（LMP）琼脂糖:添加琼脂糖至100毫升的E30.8克LMP和热，直到完全溶解。趁热等分试样1毫升LMP琼脂糖成在37℃加热块1.5毫升离心管。在LMP琼脂糖将保持在37℃熔融。添加40微升4毫克/毫升（25倍），pH 7.0的三卡因以每次1毫升等分试样的LMP琼脂糖在37℃。注意:如果使用色素应变工作，加入20微升0.15％（50倍）PTU至每1ml等分。 <ol><li>存储剩余的LMP琼脂糖在密封50ml试管数月，在4℃。 </li></ol></li><li>麻醉联体胚胎加入1毫升4毫克/毫升（25倍），pH 7.0的三卡因被成像到25毫的E3的胚胎已经英寸</li><li>轻轻地摇晃培养皿，使胚胎会在中间池。然后，用宽嘴塑料移液管吸取了在少液尽可能胚胎。转动吸管直立并使得它们沉淀在吸移管的最底部轻轻弹起胚胎。 <ol><li>与胚胎在移液管的底部，由移液管尖轻轻触摸到琼脂糖的表面将它们转移到LMP琼脂糖1毫升等分试样。避免转移多余的液体，因为这会稀释琼脂糖。 </li></ol></li><li>从吸管排出多余液体后，用吸管琼脂糖内轻轻混合胚胎，然后用吸管给所有的琼脂糖和胚胎转移到一个玻璃底的井（1.5号盖玻片），6-孔板中。 注意:确保胚胎转移到成像板后丢弃移液器。残余琼脂糖将设置吸移管内部，renderin克它无效的进一步使用。相反，使用新的移液管各一次。 </li><li>下一个立体镜，可以使用固定在木材处理取笑针的端部的凝胶加载枪头胚胎向下朝向护罩玻璃定位（以确保它们是显微镜物镜的工作距离内），并在所需的取向成像。 注:不断重新定位，直到琼脂糖完全建立。照顾根据该一对胚胎装入联体胚胎是将被成像。鉴于联体胚胎的随机取向，对于一些对可能难以对图像两者胚胎。在这种情况下，回收利用镊子和宽嘴塑料移液管从琼脂糖胚胎，然后从不同的角度重新装入成像。 </li><li>一旦琼脂糖已设置，覆盖2 - 3毫升E3的补充有三卡因（和PTU如有必要）。 </li><li>图像用倒置的宽视场落射荧光，拉斯胚胎ER-扫描共聚焦，或旋转盘共聚焦显微镜。选择最适合于期望的成像目标。换一个全胚胎区​​，使用4倍的目标。选择较高的倍率，比如20倍的目标，将图像特定组织16，19。 注意:如果使用正置显微镜，安装在盖玻片LMP琼脂糖（类似于上文）。倒置玻璃盖玻片上具有真空润滑脂填充有相同的E3-三卡因-PTU 16,19的环的玻璃显微镜载片。 </li><li>过程中获得自由使用图像分析软件包，如NIH图像的J / FIJI 16，19的图像。 注:计数细胞的数量和量化动态，如细 ​​胞迁移和细胞分裂使用分割和跟踪算法16，19。 </li></ol>

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Physiol. 145 (2), 336-352 (1959).</li><li>Goldman, D. C., Bailey, A. S., Pfaffle, D. L., Al Masri, A., Christian, J. L., Fleming, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP4+regulates+the+hematopoietic+stem+cell+niche.">BMP4 regulates the hematopoietic stem cell niche.</a> Blood. 114 (20), 4393-4401 (2009).</li><li>Dieterlen-Li&#232;vre, F., Martin, C., Beaupain, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quail-chick+chimaeras+and+parabionts:+several+new+models+to+investigate+early+developmental+events+in+the+haemopoietic+system.">Quail-chick chimaeras and parabionts: several new models to investigate early developmental events in the haemopoietic system.</a> Folia Biol (Praha). 25 (5), 293-295 (1979).</li><li>Demy, D. L., Ranta, Z., Giorgi, J. 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Methods. 10 (3), 256-258 (2013).</li><li>Anderson, 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=Hematopoietic+stem+cells+develop+the+absence+of+endothelial+cadherin+5+expression.">Hematopoietic stem cells develop the absence of endothelial cadherin 5 expression.</a> Blood. 126 (26), 2811-2820 (2015).</li><li>Murayama, E., Sarris, M., Redd, M., Le Guyader, D., Vivier, C., Horsley, W., Trede, N., Herbomel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NACA+deficiency+reveals+the+crucial+role+of+somite-derived+stromal+cells+in+haematopoietic+niche+formation.">NACA deficiency reveals the crucial role of somite-derived stromal cells in haematopoietic niche formation.</a> Nat Commun. 28 (6), 8375 (2015).</li><li>Shah, D. I., 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=Mitochondrial+Atpif1+regulates+haem+synthesis+in+developing+erythroblasts.">Mitochondrial Atpif1 regulates haem synthesis in developing erythroblasts.</a> Nature. 491 (7425), 608-612 (2012).</li><li>Tamplin, O. J., 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=Hematopoietic+stem+cell+arrival+triggers+dynamic+remodeling+of+the+perivascular+niche.">Hematopoietic stem cell arrival triggers dynamic remodeling of the perivascular niche.</a> Cell. 160 (1-2), 241-252 (2015).</li><li>Ad&#225;m, A., B&#225;rtfai, R., Lele, Z., Krone, P. H., Orb&#225;n, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat-inducible+expression+of+a+reporter+gene+detected+by+transient+assay+in+zebrafish.">Heat-inducible expression of a reporter gene detected by transient assay in zebrafish.</a> Exp. Cell Res. 256 (1), 282-290 (2000).</li></ol>

The authors declare that they have no conflict of interest.

联体融合已调查在成年鼠模型和鸡胚10-14候选基因的细胞功能的有力工具。最近，囊胚融合方法已用于产生连体斑马鱼胚胎15所述。在本协议中，基于视频的教程用于演示和更好地描述的方法为，以研究在造血发育感兴趣的候选基因的时空，细胞 - 本征的，和细胞外的角色创建不同遗传背景的连体斑马鱼的胚胎超越。 本文中所描述的方法，最近被用于跟踪的HSC...

野生型和转基因动物的遗传马赛克（嵌合体）的创建是调查细胞内在与候选基因1-6的细胞外功能的完善和经典的策略。在斑马鱼囊胚移植已被广泛使用，以产生用于细胞自治7-9的研究嵌合胚胎。取决于感兴趣的组织，但是，它可以是具有挑战性的供体细胞可预测定位到期望的组织（ 例如，血液）1-3，7-9。鼠标遗传学家早就采用连体手术的方法来产生连体生物与共享循环10-14。由于联体动物都有一个共同的血液，即来自一种动物具有不同遗传背景的其他动物的细胞的细胞之间的相互作用，可以研究10-16。近日，Karima Kissa小组优雅证明CRE的能力连体吃斑马鱼胚胎，然后用这个系统来研究造血干祖细胞（HSPC）的迁移15。此外，斑马鱼联体最近用于研究内皮钙5的造血干细胞（HSC）的出现和迁移，16的作用和斑马鱼发育17中，研究中的HSPC利基基质细胞的作用。 不像小鼠，斑马鱼的胚胎是透明的，允许联体发育过程中的细胞的直接可视化，使得系统唯一地强大。斑马鱼中的联体的效用，然而，由一个陡峭的学习曲线，并在胚胎细腻手术连体限制可以没有详细的说明和视觉示范技术上的挑战。该协议的目标是提供一套明确的，一步一步的指示陪如何产生连体斑马鱼胚胎为研究基于视频的教程颞，细胞 - 本征的，或者一个候选基因（s）的显影囊胚的手术融合的细胞外功能。重要的改进和提高parabiont生存和新的实验性应用程序的其他建议包括在内。

<table border="0" cellpadding="0" cellspacing="0" width="686">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Methyl cellulose</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:167px;">M0387</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Individual components for E3/HCR:</td>
			<td></td>
			<td style="width:167px;">For 1L: 14,61g NaCl, 0,63g KCl, 2,43g CaCl2-2H2O, 1,99g MgSO4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl (Sodium chloride)</td>
			<td style="width:87px;">Sigma-Aldrich</td>
			<td style="width:167px;">S9888</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl (Potassium chloride)</td>
			<td>Sigma-Aldrich</td>
			<td style="width:167px;">P9541</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2 (Calcium chloride dihydrate)</td>
			<td>Sigma-Aldrich</td>
			<td style="width:167px;">223506</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4 (Magnessium sulfate heptahydrate)</td>
			<td>Sigma-Aldrich</td>
			<td style="width:167px;">230391</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hepes (1M ) buffer solution</td>
			<td style="width:87px;">ThermoFisher</td>
			<td>15630-080</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Name</td>
			<td style="width:87px;">Company</td>
			<td style="width:167px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibiotics:</td>
			<td style="width:87px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pen/Strep</td>
			<td style="width:87px;">gibco by Life technologies</td>
			<td>15140-120</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ampicilin sodium salt</td>
			<td style="width:87px;">Sigma Life Science</td>
			<td style="width:167px;">A0166</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kanamycin sulfate from Streptomyces kanamyceticus</td>
			<td style="width:87px;">Sigma Life Science</td>
			<td style="width:167px;">K1377</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Name of Reagent/ Equipment</td>
			<td style="width:87px;">Company</td>
			<td style="width:167px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 mg/ml Pronase from Streptomyces griseus</td>
			<td style="width:87px;">Roche</td>
			<td style="width:167px;">11459643001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">capillary glass used for needles (Capillary Glass &#38; Filaments)</td>
			<td style="width:87px;">Sutter Instrument</td>
			<td style="width:167px;">ITEM#: BF 100-50-10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Teasing needles with wooden handles</td>
			<td style="width:87px;">Fisher Scientific</td>
			<td>S07894</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass Pasteur pipettes</td>
			<td style="width:87px;">Fisherbrand</td>
			<td style="width:167px;">13-678-20A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 mL pipette pump (green) (Pipette Pump Pipettor)</td>
			<td style="width:87px;">Novatech International</td>
			<td style="width:167px;">F37898-0000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mm diameter/ 20 mm deep plastic petri dishes (PETRI DISH, 100/20 MM, PS, CLEAR, WITH VENTS, 
			HEAVY DESIGN, 15 PCS./BAG )</td>
			<td>Greiner Bio-one</td>
			<td>664102</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextran, Cascade Blue, 10,000 MW, Anionic, Lysine Fixable</td>
			<td>ThermoFisher</td>
			<td>D-1976</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PTU. working stock is 0.003% (50X is 0.15%). for 500ml, 0.75 g N-Phenylthiourea</td>
			<td style="width:87px;">Sigma-Aldrich</td>
			<td style="width:167px;">P7629</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tricaine (powder) (Tricaine Methanesulfonato, Tricaine-S)</td>
			<td style="width:87px;">Western Chemical Inc</td>
			<td style="width:167px;">MS 222</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LMP agarose (Ultrapure LMP agarose)</td>
			<td style="width:87px;">Invitrogen</td>
			<td style="width:167px;">16520100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">plastic transfer pipette (just the wide ended one I think)</td>
			<td style="width:87px;">Fisherbrand</td>
			<td style="width:167px;">137115AM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass-bottom 6-well plates used for imaging</td>
			<td style="width:87px;">MatTek</td>
			<td>P06G1.5-20-F</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">plastic western gel loading tip fixed on the end of a wood-handled dissecting needle (GELoader tips)</td>
			<td style="width:87px;">Eppendorf</td>
			<td style="width:167px;">22351656</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">glass cover slips, slides and vacuum grease if mounting for an upright microscope:</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Vaccum grease ( Dow Corning&#174; high-vacuum silicone grease 
			colorless, weight 5.3 oz (tube) )</td>
			<td style="width:87px;">Dow Corning</td>
			<td>Z273554</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Glass cover slips</td>
			<td style="width:87px;">Corning Life Sciences</td>
			<td style="width:167px;">2960-244</td>
		</tr>
	</tbody>
</table>

generation of parabiotic zebrafish embryos by surgical fusion of developing blastulae

不同的遗传背景的两种动物的手术间生态创建了一个独特的场景来研究细胞内在与感兴趣的候选基因，细胞的迁徙行为，并在不同的遗传设置分泌信号，细胞外源性的角色。由于联体动物有着共同的循环，从一个动物的血液或血源性因素将与其合作伙伴，反之亦然交换。因此，从一个遗传背景的细胞和分子因素可在第二遗传背景的情况下进行研究。成年小鼠的联体已被广泛用于研究衰老，癌症，糖尿病，肥胖，和大脑的发育。最近，斑马鱼胚胎的联体已被用于研究造血发育生物学。与此相反的小鼠，斑马鱼胚胎的透明特性允许细胞的直接可视化的背景下连体，使其成为调查为f的独特而强大的方法undamental细胞和分子机制。这种技术的效用，但是，通过用于产生连体斑马鱼胚胎陡峭的学习曲线限定。这个协议提供了关于如何手术融合不同的遗传背景两个斑马鱼胚胎的囊胚调查感兴趣的候选基因的作用的工序一步方法。此外，联体斑马鱼的胚胎是耐受热冲击，使得基因表达可能的时间控制。该方法不需要复杂的设置和对胚胎发育过程中研究体内细胞迁移，命运说明书中，并分化广阔的应用领域。

与先前发表的研究15一致，斑马鱼胚胎的成功联体融合取决于两个胚胎的分期和取向和甲基纤维素的浓度。只需几个简单的，廉价的工具，产生了手术融合发展囊胚，增长到连体胚胎共享的循环。这些工具包括一个修改巴斯德吸管，10毫升吸管泵，以及其中使用单独的或与一种凝胶装载尖端或玻璃微注射针通过实验室膜固定到端（ 图1）木材处理取笑针...

Watch this Scientific Journal Video about Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae at JoVE.com

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

联体斑马鱼胚胎一代发展囊胚的手术融合

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

generation-parabiotic-zebrafish-embryos-surgical-fusion-developing

Research

JoVE Journal

Developmental Biology

11.4K 次观看.  Boston Children’s Hospital. This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

视频: Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

Mechanical Vessel Injury in Zebrafish Embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

在斑马鱼胚胎的机械血管损伤

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

科研

发育生物学

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

分离和斑马鱼胚胎单细胞的表征

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish embryos has been implemented. This technique takes advantage of the selectivity and sensitivity conferred by affinity labeling of a given receptor by its ligand with the specificity of the immunoprecipitation. We have used AFLIP to detect the type III TGF-&#946; receptor (TGFBR3), also know as betaglycan, during early zebrafish development. AFLIP was instrumental in validating the efficacy of a TGFBR3 morphant zebrafish phenotype. In the first step, embryo protein extracts are prepared and used to generate 125I-TGF-&#946;2-TGFBR3 complexes that are purified by immunoprecipitation. Later, these complexes are covalently cross-linked and revealed using SDS-PAGE separation and autoradiography detection. This technique requires the availability of a labeled ligand for, and a specific antibody against, the receptor to be detected, and shall be easily adapted to identify any growth factor or cytokine receptor that meets these requirements.

A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

从斑马鱼胚胎内源性受体亲和标记检测

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, specifically the generation of homozygous gynogenetic diploids is useful in genetic analysis because it allows the direct production of homozygotes from a single heterozygous mother. This article describes a modified protocol for ploidy duplication based on a heat pulse during the first cell cycle, Heat Shock 2 (HS2). Through inhibition of centriole duplication, this method results in a precise cell division stall during the second cell cycle. The precise one-cycle division stall, coupled to unaffected DNA duplication, results in whole genome duplication. Protocols associated with this method include egg and sperm collection, UV treatment of sperm, in vitro fertilization and heat pulse to cause a one-cell cycle division delay and ploidy duplication. A modified version of this protocol could be applied to induce ploidy changes in other animal species.

A modified protocol for ploidy manipulation uses a heat shock to induce a one-cycle stall in cytokinesis in the early embryo. This protocol is demonstrated in the zebrafish but may be applicable to other species.

斑马鱼胚胎的倍性操作与热休克治疗2

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

生物传感运动神经元膜电位在活斑马鱼胚胎

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

诱导生活青蛙和斑马鱼胚胎缺氧

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell lineages that comprise mature blood. Isolation and identification of these HSPCs is difficult because they are defined ex post facto; they can only be defined after their differentiation into specific cell lineages. Over the past few decades, the zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop ex utero, and by 48 h post-fertilization (hpf) have generated definitive HSPCs. Assays to assess HSPC differentiation and proliferation capabilities have been developed, utilizing transplantation and subsequent reconstitution of the hematopoietic system in addition to visualizing specialized transgenic lines with confocal microscopy. However, these assays are cost prohibitive, technically difficult, and time consuming for many laboratories. Development of an in vitro model to assess HSPCs would be cost effective, quicker, and present fewer difficulties compared to previously described methods, allowing laboratories to quickly assess mutagenesis and drug screens that affect HSPC biology. This novel in vitro assay to assess HSPCs is performed by plating dissociated whole zebrafish embryos and adding exogenous factors that promote only HSPC differentiation and proliferation. Embryos are dissociated into single cells and plated with HSPC-supportive colony stimulating factors that cause them to generate colony forming units (CFUs) that arise from a single progenitor cell. These assays should allow more careful examination of the molecular pathways responsible for HSPC proliferation, differentiation, and regulation, which will allow researchers to understand the underpinnings of vertebrate hematopoiesis and its dysregulation during disease.

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

Here, we present a simple method to quantitate hematopoietic stem and progenitor cells (HSPCs) in embryonic zebrafish. HSPCs from dissociated zebrafish are plated in methylcellulose with supportive factors, differentiating into mature blood. This allows the detection of blood defects and allows drug screening to be easily conducted.

定量造血干和祖细胞 (HSPCs) 在斑马鱼胚胎发育中的体外试验研究

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

斑马鱼早期胚胎和肿瘤细胞电活动的可视化研究

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Surgical Size Reduction of Zebrafish for the Study of Embryonic Pattern Scaling

In the developmental process, embryos exhibit a remarkable ability to match their body pattern to their body size; their body proportion is maintained even in embryos that are larger or smaller, within certain limits. Although this phenomenon of scaling has attracted attention for over a century, understanding the underlying mechanisms has been limited, owing in part to a lack of quantitative description of developmental dynamics in embryos of varied sizes. To overcome this limitation, we developed a new technique to surgically reduce the size of zebrafish embryos, which have great advantages for in vivo live imaging. We demonstrate that after balanced removal of cells and yolk at the blastula stage in separate steps, embryos can quickly recover under the right conditions and develop into smaller but otherwise normal embryos. Since this technique does not require special equipment, it is easily adaptable, and can be used to study a wide range of scaling problems, including robustness of morphogen mediated patterning.&#160;

Here, we describe a method for reducing the size of zebrafish embryos without disrupting normal developmental processes. This technique enables the study of pattern scaling and developmental robustness against size change.

斑马鱼手术尺寸缩小术中胚胎形态缩放的研究

In the developmental process, embryos exhibit a remarkable ability to match their body pattern to their body size; their body proportion is maintained ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

斑马鱼胚胎和幼虫的整个山免疫性化学

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...