The authors thank Drs. Hukriede and Tsang for discussions. M.K. was supported by an NIH training grant (T32EB001026). This work was supported in part by grants from the American Liver Foundation, the March of Dimes Foundation (5-FY12-39), and the NIH (DK101426) to D.S.

斑马鱼根据标准程序提出并饲养;实验经匹兹堡机构动物护理和使用委员会的大学。 1.准备胚胎/幼虫<ol><li>要定时进行交配，建立成年男性和女性的Tg（fabp10a:CFP-NTR）S931半合子或纯合子鱼O / N，把它们之间的分隔。交配时需要删除此分压器的第二天早晨。用细塑料滤网紧张的水收集胚胎。 </li><li>打开过滤器倒置和冲洗网面蛋水的细流从洗瓶分配。转移100胚胎成100毫米培养皿用25毫升水蛋的。保持不超过100元的胚胎培养皿中，并提高它们在28℃。 </li><li>为了抑制色素沉着，添加苯基硫脲嘧啶（PTU）进入前10小时受精后（HPF）的培养皿，并提高Ëmbryos /幼虫，直到80高倍视野。 PTU的最终浓度为0.2毫摩尔。注意:PTU是有毒的。使用按照适当的处理方针。 </li><li>通过加入3-氨基苯甲酸乙酯（三卡因），在0.016％的最终浓度，进培养皿麻醉幼虫。然后，用落射荧光显微镜，排序CFP阳性幼虫。使用幼虫具有相似肝脏大小并丢弃那些具有更小或更大的肝脏。 注意:任何CFP阳性幼体，无论强度，可以使用，因为在肝切除的半合子和纯合子幼虫之间的程度没有差别。注意:三卡因是有毒的。使用按照适当的处理方针。 </li><li>删除包含三卡因鸡蛋水中加辅以0.2毫米PTU鸡蛋水。保持胚胎/幼虫在28℃。 </li></ol> 2.治疗甲硝唑<ol><li>在1溶解0.171克甲硝唑粉准备新鲜的10毫米甲硝唑液00毫升蛋水补充有0.2％DMS​​O和0.2mM的PTU。以完全溶解甲硝唑，混合溶液在室温下快速搅拌下进行20-30分钟。为了帮助溶解甲硝唑和提升甲硝唑渗透到幼虫，准备甲硝唑时添加DMSO。注意:长期接触或甲硝唑的浓度增加是有毒的。使用按照适当的处理方针。 </li><li>通过转移幼虫所需数目为两个不同百毫米培养皿或多孔板幼虫到对照组和实验组中分离出来。对于实验组，保持在10mM甲硝唑溶液幼虫;为对照组，它们保存在含0.2％DMS​​O的蛋水。 </li><li>覆盖板或含有用铝箔甲硝唑溶液，以防止光灭活甲硝唑的培养皿，并培养在28℃。甲硝唑治疗的持续时间依赖于幼虫期和转基因系。 </li><li>观察类似的严重肝切除<eM&gt;的Tg（fabp10a:CFP-NTR）线，对待同来自3.5天受精后（DPF）的甲硝唑溶液中的幼虫（36小时的持续时间）或5 DPF（为24小时的持续时间）。 </li></ol>胆道驱动肝再生3.分析<ol><li>在消融期间结束，取出从板的甲硝唑溶液。加入25毫升蛋水冲洗的甲硝唑治疗的幼虫。旋流板的2-3倍幼虫废弃卵子前水冲洗。重复三次，然后将其浸入幼虫再次在蛋水。避免心脏水肿和幼虫死亡中甲硝唑处理的幼虫中，添加三卡因的浓度较低，在0.008％的最终浓度，以固定的幼虫。 </li><li>对于肝脏的分析，考察甲硝唑治疗幼虫的肝脏大小的落射荧光显微镜与CFP过滤器下。评估基于相对肝脏大小肝细胞消融级别:大（非烧蚀; 0-10％的幼虫），中（部分地烧蚀; 10-20％），以及非常小的（完全烧蚀; 80-90％）。 </li><li>删除非消融或部分肝脏切除的幼虫。仅保持幼虫一个微小肝脏，其表示严重肝切除。在再生0小时的时间点（R0H）肝脏分析，收获甲硝唑冲洗和CFP排序后的幼虫。否则，保持水蛋排序幼虫辅以0.2毫米PTU直至准备收获。 </li><li>转移收获幼体至1.5 ml微量离心管中，并用3％甲醛在PEM缓冲定影溶液代替鸡蛋水。孵育在旋转器上O / N在4℃下在管。 </li><li>替换用1ml PBSDT的固定溶液和旋转载转子幼虫5分钟的管。 </li><li>固定幼虫转移到含有PBSDT和使用镊子100毫米培养皿，在解剖显微镜下手工除去卵黄和胸鳍。传送多达TP 20解剖幼虫到1.5毫升管。 </li> &lt;利&gt;替换PBSDT溶液与1ml封闭溶液，并摇动试管旋转器上在室温搅拌2小时。 </li><li>更换封闭液用100μl阻断含有初级抗体溶液。慢慢的岩石上一肩O / N在4℃管。 </li><li>除去第一抗体溶液，并用PBSDT 5次，每次10分钟。再加入100微升封闭液含二级抗体和岩石它在旋转器上在室温2小时的。 </li><li>摇动旋转器上洗5次PBSDT为每次10分钟，在室温。 </li><li>横向东方幼虫在显微镜幻灯片，加入一滴安装介质。仔细包括他们用玻璃盖，密封玻璃盖用指甲抛光。现在，它已经准备好共聚焦显微镜。 </li></ol>

<ol>
	<li>Riehle, K. J., Dan, Y. Y., Campbell, J. S., Fausto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+concepts+in+liver+regeneration.">New concepts in liver regeneration.</a> J Gastroen Hepatol. 26, 203-212 (2011).</li><li>Fausto, N., Campbell, J. 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+role+of+hepatocytes+and+oval+cells+in+liver+regeneration+and+repopulation.">The role of hepatocytes and oval cells in liver regeneration and repopulation.</a> Mechanisms of Development. 120, 117-130 (2003).</li><li>Duncan, A. W., Dorrell, C., Grompe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+Cells+and+Liver+Regeneration.">Stem Cells and Liver Regeneration.</a> Gastroenterology. 137, 466-481 (2009).</li><li>Michalopoulos, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+regeneration:+Alternative+epithelial+pathways.">Liver regeneration: Alternative epithelial pathways.</a> Int J Biochem Cell B. 43, 173-179 (2011).</li><li>Falkowski, 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=Regeneration+of+hepatocyte+'buds'+in+cirrhosis+from+intrabiliary+stem+cells.">Regeneration of hepatocyte 'buds' in cirrhosis from intrabiliary stem cells.</a> Journal of hepatology. 39, 357-364 (2003).</li><li>Yoon, S. 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=Epithelial+Cell+Adhesion+Molecule+(EpCAM)+Marks+Hepatocytes+Newly+Derived+from+Stem/Progenitor+Cells+in+Humans.">Epithelial Cell Adhesion Molecule (EpCAM) Marks Hepatocytes Newly Derived from Stem/Progenitor Cells in Humans.</a> Hepatology. 53, 964-973 (2011).</li><li>Michalopoulos, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+regeneration.">Liver regeneration.</a> J Cell Physiol. 213, 286-300 (2007).</li><li>Michalopoulos, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+Regeneration+after+Partial+Hepatectomy+Critical+Analysis+of+Mechanistic+Dilemmas.">Liver Regeneration after Partial Hepatectomy Critical Analysis of Mechanistic Dilemmas.</a> Am J Pathol. 176, 2-13 (2010).</li><li>Yanger, 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=Adult+hepatocytes+are+generated+by+self-duplication+rather+than+stem+cell+differentiation.">Adult hepatocytes are generated by self-duplication rather than stem cell differentiation.</a> Cell stem cell. 15, 340-349 (2014).</li><li>Schaub, J. R., Malato, Y., Gormond, C., Willenbring, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+against+a+Stem+Cell+Origin+of+New+Hepatocytes+in+a+Common+Mouse+Model+of+Chronic+Liver+Injury.">Evidence against a Stem Cell Origin of New Hepatocytes in a Common Mouse Model of Chronic Liver Injury.</a> Cell reports. 8, 933-939 (2014).</li><li>Dovey, 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=Topoisomerase+II+alpha+is+required+for+embryonic+development+and+liver+regeneration+in+zebrafish.">Topoisomerase II alpha is required for embryonic development and liver regeneration in zebrafish.</a> Molecular and cellular biology. 29, 3746-3753 (2009).</li><li>Goessling, W., 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=APC+mutant+zebrafish+uncover+a+changing+temporal+requirement+for+wnt+signaling+in+liver+development.">APC mutant zebrafish uncover a changing temporal requirement for wnt signaling in liver development.</a> Developmental Biology. 320, 161-174 (2008).</li><li>Sadler, K. C., Krahn, K. N., Gaur, N. A., Ukomadu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+growth+in+the+embryo+and+during+liver+regeneration+in+zebrafish+requires+the+cell+cycle+regulator,+uhrf1.">Liver growth in the embryo and during liver regeneration in zebrafish requires the cell cycle regulator, uhrf1.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 1570-1575 (2007).</li><li>Cox, A. G., 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=S-nitrosothiol+signaling+regulates+liver+development+and+improves+outcome+following+toxic+liver+injury.">S-nitrosothiol signaling regulates liver development and improves outcome following toxic liver injury.</a> Cell reports. 6, 56-69 (2014).</li><li>North, T. E., 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=PGE2-regulated+wnt+signaling+and+N-acetylcysteine+are+synergistically+hepatoprotective+in+zebrafish+acetaminophen+injury.">PGE2-regulated wnt signaling and N-acetylcysteine are synergistically hepatoprotective in zebrafish acetaminophen injury.</a> Proc Natl Acad Sci U S A. 107, 17315-17320 (2010).</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> Developmental Dynamics. 236, 1025-1035 (2007).</li><li>Pisharath, H., Rhee, J. M., Swanson, M. A., Leach, S. D., Parsons, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+ablation+of+beta+cells+in+the+embryonic+zebrafish+pancreas+using+E.+coli+nitroreductase.">Targeted ablation of beta cells in the embryonic zebrafish pancreas using E. coli nitroreductase.</a> Mech Dev. 124, 218-229 (2007).</li><li>Curado, S., Stainier, D. Y. R., Anderson, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitroreductase-mediated+cell/tissue+ablation+in+zebrafish:+a+spatially+and+temporally+controlled+ablation+method+with+applications+in+developmental+and+regeneration+studies.">Nitroreductase-mediated cell/tissue ablation in zebrafish: a spatially and temporally controlled ablation method with applications in developmental and regeneration studies.</a> Nat Protoc. 3, 948-954 (2008).</li><li>Choi, T. Y., Ninov, N., Stainier, D. Y., Shin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+conversion+of+hepatic+biliary+epithelial+cells+to+hepatocytes+after+near+total+loss+of+hepatocytes+in+zebrafish.">Extensive conversion of hepatic biliary epithelial cells to hepatocytes after near total loss of hepatocytes in zebrafish.</a> Gastroenterology. 146, 776-788 (2014).</li><li>Huang, 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=Antagonistic+interaction+between+Wnt+and+Notch+activity+modulates+the+regenerative+capacity+of+a+zebrafish+fibrotic+liver+model.">Antagonistic interaction between Wnt and Notch activity modulates the regenerative capacity of a zebrafish fibrotic liver model.</a> Hepatology. , (2014).</li><li>He, J., Lu, H., Zou, Q., Luo, L. <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+liver+after+extreme+hepatocyte+loss+occurs+mainly+via+biliary+transdifferentiation+in+zebrafish.">Regeneration of liver after extreme hepatocyte loss occurs mainly via biliary transdifferentiation in zebrafish.</a> Gastroenterology. 146, 789-800 (2014).</li><li>Sakaguchi, T. F., Sadler, K. C., Crosnier, C., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+signals+modulate+hepatocyte+apicobasal+polarization+in+zebrafish.">Endothelial signals modulate hepatocyte apicobasal polarization in zebrafish.</a> Curr Biol. 18, 1565-1571 (2008).</li><li>Ninov, N., Borius, M., Stainier, D. Y. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+levels+of+Notch+signaling+regulate+quiescence,+renewal+and+differentiation+in+pancreatic+endocrine+progenitors.">Different levels of Notch signaling regulate quiescence, renewal and differentiation in pancreatic endocrine progenitors.</a> Development. 139, 1557-1567 (2012).</li><li>Lorent, K., Moore, J. C., Siekmann, A. F., Lawson, N., Pack, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reiterative+use+of+the+notch+signal+during+zebrafish+intrahepatic+biliary+development.">Reiterative use of the notch signal during zebrafish intrahepatic biliary development.</a> Developmental dynamics : an official publication of the American Association of Anatomists. 239, 855-864 (2010).</li><li>Itoh, T., Miyajima, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+Regeneration+by+Stem/Progenitor+Cells.">Liver Regeneration by Stem/Progenitor Cells.</a> Hepatology. 59, 1617-1626 (2014).</li><li>Mathias, J. R., Zhang, Z., Saxena, M. T., Mumm, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+cell-specific+ablation+in+zebrafish+using+a+triple+mutant+of+Escherichia+coli+nitroreductase.">Enhanced cell-specific ablation in zebrafish using a triple mutant of Escherichia coli nitroreductase.</a> Zebrafish. 11, 85-97 (2014).</li><li>Stueck, A. E., Wanless, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regression+of+cirrhosis:+The+maturation+sequence+of+buds+arising+from+hepatocyte+progenitor+cells.">Regression of cirrhosis: The maturation sequence of buds arising from hepatocyte progenitor cells.</a> Hepatology. 58, 235A (2013).</li></ol>

作者宣称，他们有没有竞争的财务权益。

严重的，但不是轻微，肝细胞消融引起胆道驱动的肝脏再生。 CFP-NTR基因:因此，以下甲硝唑治疗和清洗，以检查甲硝唑处理的幼虫，其可以通过固有的CFP荧光从fabp10a进行评估的肝脏大小是重要的。由于甲硝唑处理幼虫将显示非烧蚀（0-5％）或部分烧蚀（10-20％）的肝脏的一小部分，它是必不可少的梳理和只分析幼虫具有一个非常小的肝脏，这表明严重肝切除。 <p class="jove_co...

肝脏是一个高度再生的器官。在肝再生，再生肝细胞，肝实质细胞，衍生自预先存在的肝细胞（肝细胞驱动的肝脏再生）或的BEC（胆驱动肝再生）1,2。肝损伤引起，通常预先存在肝细胞的增殖;然而，当肝细胞增殖受到损害，建筑物能源效益守则可以促进肝细胞2-4。肝脏再生的这两种模式是临床显著。在手术切除（因为肝肿瘤或活肝供体例如 ）人类肝脏的一部分的，在剩余的肝的肝细胞增殖来恢复丢失的肝脏重量。与此相反，在患有严重肝病，肝细胞增殖受到很大损害，使得的BEC或肝脏祖细胞（LPCS）似乎有助于再生肝细胞5,6。啮齿动物的2/3部分肝切除模型，其中肝细胞增殖，以恢复丢失的肝脏肿块，已经显著促进了肝细胞驱动的肝脏再生7,8当前的理解。然而，存在其中再生肝细胞主要来源于BEC中没有有效的啮齿动物模型。尽管一些啮齿类动物肝脏毒素模型导致胆道驱动肝细胞再生2-4的鉴定，在小鼠近期谱系追踪研究表明建筑物能源效益守则，以在这些模型中9,10再生肝细胞最小的贡献。一些啮齿类肝损伤模型，其中包括部分肝切除11-13和对乙酰氨基酚引起的肝损害14,15，已经被应用到斑马鱼和导致牵连在肝再生新基因或通路的鉴定。然而，肝细胞驱动的，而不是BEC驱动，肝再生发生在这些斑马鱼肝损伤模型。因此，一种新的肝损伤模型中建筑物能源效益守则广泛有助于regenerat荷兰国际集团肝细胞都需要更好地了解BEC驱动的肝脏再生。 肝细胞消融模型的总体目标描述此处被（1），以产生一个肝损伤模型，其中的BEC广泛向再生肝细胞，和（2）阐明底层BEC驱动肝脏再生的分子和细胞机制。我们假设，损伤的严重程度决定了肝再生的模式;因此，我们预测，胆驱动肝细胞再生会引发严重时肝细胞损伤。为了检验这一假设，我们开发了斑马鱼肝损伤模型通过产生一个转基因品系，Tg为（fabp10a:CFP-NTR）S931，即高表达细菌硝基还原酶（NTR）融合青色荧光蛋白（CFP）的肝细胞特异性fabp10a下子。因为NTR转换无毒前药，甲硝唑（甲硝唑），成细胞毒性药物，它烧蚀只有预期NTR-表达细胞16-18，在这种情况下，肝细胞。通过操纵甲硝唑治疗的持续时间，肝细胞烧蚀的程度可被控制。利用该模型，我们最近报道说，在严重的肝细胞损失，BEC中广泛地再生肝细胞19，其进一步通过另外两个独立的研究20,21证实产生。因此，与上述的啮齿动物和斑马鱼肝损伤模型，我们的肝切除模型是用于研究BEC驱动肝再生更有利。 本协议描述了使用斑马鱼肝切除模型进行肝再生的实验过程。这个模型将是适当用于确定底层胆道驱动肝脏再生的机制和化学筛选，以确定小分子，可以抑制或增强肝再生。

<table><tbody><tr><td>3-amino benzoic acid ethyl ester (Tricaine) powder</td><td>Sigma-Aldrich</td><td>A5040</td><td>Make 0.4% (15 mM) tricaine stock : Bring 0.4 g tricaine to 100 mL with egg water. Adjust to pH 7.&amp;nbsp;</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D8418</td><td></td></tr><tr><td>Metronidazole</td><td>Sigma-Aldrich</td><td>M1547</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8532</td><td></td></tr><tr><td>Formaldehyde</td><td>Sigma-Aldrich</td><td>252549</td><td></td></tr><tr><td>Calcium sulfate dihydrate</td><td>Sigma-Aldrich</td><td>C3771</td><td></td></tr><tr><td>N-Phenylthiourea (PTU)</td><td>Sigma-Aldrich</td><td>P7629</td><td></td></tr><tr><td>Mounting media (Vectashield)</td><td>Vector Laboratories</td><td>H-1000</td><td></td></tr><tr><td>100 x 15 mm petri dish&amp;nbsp;</td><td>Denville Scientific Inc.</td><td>M5300</td><td></td></tr><tr><td>clear nail polish</td><td>Fisher Scientific</td><td>50949071</td><td></td></tr><tr><td>fine forceps</td><td>Fine Science Tools</td><td>11251-20</td><td>Dumont #5&amp;nbsp;</td></tr><tr><td>glass slide</td><td>Fisher Scientific</td><td>12-544-1</td><td></td></tr><tr><td>glass coverslip</td><td>Fisher Scientific</td><td>12-540-A and 12-542-A</td><td>18 x 18 mm</td></tr><tr><td>zn-5 (mouse anti-Alcam)</td><td>ZIRC</td><td>zn-5</td><td>1:10 dilution</td></tr><tr><td>goat anti-Hnf4a</td><td>Santa Cruz</td><td>sc-6556 (C-19)</td><td>1:50 dilution</td></tr><tr><td>rat anti-RFP</td><td>Allele Biotechnology</td><td>ACT-CM-MRRFP10</td><td>1:300 dilution</td></tr><tr><td>AlexaFluor 647 AffiniPure Donkey Anti-goat IgG (H+L)</td><td>Jackson laboratories</td><td>705-605-147</td><td>1:500 dilution</td></tr><tr><td>AlexaFluor 647 AffiniPure Goat Anti-mouse IgG (H+L)</td><td>Invitrogen</td><td>A21240</td><td>1:500 dilution</td></tr><tr><td>Cy3 donkey anti-rat IgG</td><td>Jackson laboratories</td><td>712-165-150</td><td>1:500 dilution</td></tr><tr><td>dissecting microscope</td><td>Leica Microsystems</td><td>S6F</td><td></td></tr><tr><td>epifluorescence microscope</td><td>Leica Microsystems</td><td>M205FA</td><td></td></tr><tr><td>confocal microscope</td><td>Carl Zeiss</td><td>LSM700</td><td></td></tr><tr><td>egg water</td><td></td><td></td><td>0.3 g/L of sea salts &amp;lsquo;Instant Ocean&amp;rsquo; and 0.5 mM CaSO&lt;sub&gt;4 &lt;/sub&gt;in distilled water.&lt;br /&gt;
&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>10X PBS</td><td></td><td></td><td>25.6 g Na2HPO4&amp;middot;7H2O, 80 g NaCl, 2 g KCl, 2 g KH2PO4.&amp;nbsp; Bring to 1 L with distilled water.&amp;nbsp; Adjust pH 7.&amp;nbsp; Autoclave for 20 min at 121&amp;deg;C.</td></tr><tr><td>1X PBSDT</td><td></td><td></td><td>0.2% Triton X-100 and 0.2% DMSO in 1 X PBS.</td></tr><tr><td>PEM buffer</td><td></td><td></td><td>30.2 g PIPES, 761 mg EGTA, 1 mM MgSO4.&amp;nbsp; Bring to 1 L with distilled water.&amp;nbsp; Adjust pH 7.</td></tr><tr><td>Blocking solution</td><td></td><td></td><td>Mix 1 ml of heat-inactivated horse serum with 9 ml of 1X PBSDT.</td></tr></tbody></table>

hepatocyte-specific ablation in zebrafish to study biliary-driven liver regeneration

肝脏有很大的再生能力。 hepatocyte-或胆道驱动肝细胞再生:肝细胞，肝实质细胞，可以以两种方式之一再生。在肝细胞驱动的肝脏再生，再生肝细胞是从先前存在的肝细胞衍生的，而在胆汁驱动再生，再生肝细胞是从胆管上皮细胞（BEC中）而得。对于肝细胞驱动的肝脏再生，有优秀的啮齿动物模型已经显著有助于肝脏再生的目前了解。但是，没有这样的啮齿动物模型存在胆道驱动肝再生。我们最近报告了斑马鱼肝损伤模型中的BEC广泛引起肝细胞严重时肝细胞的损失。在此模型中，肝细胞是专门由药理学手段消融。在这里，我们将详细介绍的方法来烧蚀肝细胞并分析BEC驱动肝再生过程。此肝细胞特异性消融模型可以进一步用于发现的胆驱动肝脏再生潜在的分子和细胞机制。此外，这些方法可应用于化学筛选，以确定小分子增强或抑制肝再生。

甲硝唑治疗36小时（A36h，A代表消融）从3.5到5旦显着降低肝脏大小（ 图1B）。甲硝唑冲洗后，认为是肝再生（R），强fabp10a的开头:CFP-NTR和fabp10a:30小时（ 图1B）内的红色荧光蛋白的表达又出现了。肝内胆管网络的评估，ALCAM的表达中存在的BEC中22的膜，最初折叠而重新设立在54小时后的冲洗（R54h）（ 图1C）。这些数据表明，肝再生和...

Watch this Scientific Journal Video about Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration at JoVE.com

Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

肝细胞特异性消融在斑马鱼研究胆驱动肝再生

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or ...

hepatocyte-specific-ablation-zebrafish-to-study-biliary-driven-liver

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Unit 1

Regeneration and Repair

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8.5K 次观看.  University of Pittsburgh. To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

视频: Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

肾再生的成年斑马鱼庆大霉素性损伤

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

科研

发育生物学

Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough understanding of the molecular and cellular mechanisms involved requires experimental tools that allow for inducible, tissue-specific manipulation of gene expression or signaling pathways. Therefore, we and others have recently adapted the TetON system for use in zebrafish. The TetON system facilitates temporally and spatially-controlled gene expression and we have recently used this tool to probe for tissue-specific functions of Wnt/beta&#8211;catenin signaling during zebrafish tail fin regeneration. Here we describe the workflow for using the TetON system to achieve inducible, tissue-specific gene expression in the adult regenerating zebrafish tail fin. This includes the generation of stable transgenic TetActivator and TetResponder lines, transgene induction and techniques for verification of tissue-specific gene expression in the fin regenerate. Thus, this protocol serves as blueprint for setting up a functional TetON system in zebrafish and its subsequent use, in particular for studying fin regeneration.

Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.

使用提顿体系的研究斑马鱼再生的分子机制

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.

乙醇引起的肝纤维化模型的斑马鱼发展研究祖细胞介导的​​肝细胞的再生

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

Surgical Ablation Assay for Studying Eye Regeneration in Planarians

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their abundant pluripotent stem cell population and ability to regenerate all cell and tissue types after injuries that would be catastrophic for most animals. Recently, planarians have gained popularity as a model for eye regeneration. Their ability to regenerate the entire eye (comprised of two tissue types: pigment cells and photoreceptors) allows for the dissection of the mechanisms regulating visual system regeneration. Eye ablation has several advantages over other techniques (such as decapitation or hole punch) for examining eye-specific pathways and mechanisms, the most important of which is that regeneration is largely restricted to eye tissues alone. The purpose of this video article is to demonstrate how to reliably remove the planarian optic cup without disturbing the brain or surrounding tissues. The handling of worms and maintenance of an established colony is also described. This technique uses a 31 G, 5/16-inch insulin needle to surgically scoop out the optic cup of planarians immobilized on a cold plate. This method encompasses both single and double eye ablation, with eyes regenerating within 1-2 weeks, allowing for a wide range of applications. In particular, this ablation technique can be easily combined with pharmacological and genetic (RNA interference) screens for a better understanding of regenerative mechanisms and their evolution, eye stem cells and their maintenance, and phototaxic behavioral responses and their neurological basis.

This protocol shows how to consistently excise planarian eyes (optic cups) without disturbing surrounding tissues. Using an insulin needle and syringe, either one or both eyes can be ablated to facilitate investigations into the mechanisms regulating eye regeneration, the evolution of visual regeneration, and the neural basis of light-induced behavior.

手术消融分析在研究涡虫再生眼

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their ...

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related morbidity and mortality and affects more than 2 million people in the United States. A better understanding of the cellular and molecular mechanisms underlying alcohol-induced liver injury is crucial for developing effective treatment for ALD. Zebrafish larvae exhibit hepatic steatosis and fibrogenesis after just 24 h of exposure to 2% ethanol, making them useful for the study of acute alcoholic liver injury. This work describes the procedure for acute ethanol treatment in zebrafish larvae and shows that it causes steatosis and swelling of the hepatic blood vessels. A detailed protocol for Hematoxylin and Eosin (H&#38;E) staining that is optimized for the histological analysis of the zebrafish larval liver, is also described. H&#38;E staining has several unique advantages over immunofluorescence, as it marks all liver cells and extracellular components simultaneously and can readily detect hepatic injury, such as steatosis and fibrosis. Given the increasing usage of zebrafish in modeling toxin and virus-induced liver injury, as well as inherited liver diseases, this protocol serves as a reference for the histological analyses performed in all these studies.

This protocol describes histological analyses of the livers from zebrafish larvae that have been treated with 2% ethanol for 24 h. Such acute ethanol treatment results in hepatic steatosis and swelling of the hepatic vasculature.

斑马鱼急性酒精性肝损伤的组织学分析

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related ...

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

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

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

斑马鱼非酒精性脂肪肝相关肝细胞癌早期进展期间的肝脏微环境分析

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

癌症研究

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

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

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

使用光明场显微镜定量拉瓦尔斑马鱼的肝脏大小

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

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

小鼠肝脏中细胞类型特异性基因表达分析

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...

生物学

Partial Hepatectomy in Adult Zebrafish

Liver failure is one of the leading causes of death worldwide, and mortality from chronic liver disease is rising sharply in the United States. Healthy livers are capable of regenerating from toxic damage, but in advanced liver disease, the natural ability of the liver to regenerate is impaired. Zebrafish have emerged as a powerful experimental system for studying regeneration. They are an ideal model for studying liver regeneration from partial hepatectomy, a procedure with direct clinical relevance in which part of the liver is surgically removed, leaving the rest intact. There is no standard protocol for partial hepatectomy; previous studies using this model have used slightly different protocols and reported disparate results. Described here is an efficient, reproducible protocol for performing a partial hepatectomy in adult zebrafish. We use this technique to demonstrate that zebrafish are capable of epimorphic regeneration of the resected lobe. This protocol can be used to further interrogate the mechanisms required for liver regeneration in zebrafish.

This protocol describes the procedure for removing the ventral lobe of the liver in adult zebrafish to enable the study of liver regeneration.

成人斑马鱼的部分肝切除术

Liver failure is one of the leading causes of death worldwide, and mortality from chronic liver disease is rising sharply in the United States. Healthy ...

Targeted Liver Ablation: Inducing Hepatocyte Death with Metronidazole in Transgenic Zebrafish Larvae

Source: Choi T.Y., et al. <a href="https://www.jove.com/v/52785/" target="_blank">Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration</a>. J. Vis. Exp. (2015)
The article describes targeted liver ablation, a method for inducing hepatocyte death with metronidazole in transgenic zebrafish larvae. The sample protocol describes the process for generating, manipulating and assessing targeted liver ablation in zebrafish.

靶向肝脏消融术:甲硝唑在转基因斑马鱼幼虫中诱导肝细胞死亡

Source: Choi T.Y., et al. Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration. J. Vis. Exp. (2015)
The article describes ...

肝细胞特异性消融在斑马鱼研究胆驱动肝再生

摘要

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此视频中的章节

肾再生的成年斑马鱼庆大霉素性损伤

使用提顿体系的研究斑马鱼再生的分子机制

乙醇引起的肝纤维化模型的斑马鱼发展研究祖细胞介导的肝细胞的再生

手术消融分析在研究涡虫再生眼

斑马鱼急性酒精性肝损伤的组织学分析

斑马鱼非酒精性脂肪肝相关肝细胞癌早期进展期间的肝脏微环境分析

使用光明场显微镜定量拉瓦尔斑马鱼的肝脏大小

小鼠肝脏中细胞类型特异性基因表达分析

成人斑马鱼的部分肝切除术

靶向肝脏消融术:甲硝唑在转基因斑马鱼幼虫中诱导肝细胞死亡

肝细胞特异性消融在斑马鱼研究胆驱动肝再生

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肾再生的成年斑马鱼庆大霉素性损伤

使用提顿体系的研究斑马鱼再生的分子机制

乙醇引起的肝纤维化模型的斑马鱼发展研究祖细胞介导的​​肝细胞的再生

手术消融分析在研究涡虫再生眼

斑马鱼急性酒精性肝损伤的组织学分析

斑马鱼非酒精性脂肪肝相关肝细胞癌早期进展期间的肝脏微环境分析

使用光明场显微镜定量拉瓦尔斑马鱼的肝脏大小

小鼠肝脏中细胞类型特异性基因表达分析

成人斑马鱼的部分肝切除术

靶向肝脏消融术:甲硝唑在转基因斑马鱼幼虫中诱导肝细胞死亡

乙醇引起的肝纤维化模型的斑马鱼发展研究祖细胞介导的肝细胞的再生