我们非常感谢LaRéunion大学的杜蕾斯大学（DUN）编辑视频（特别是Jean-FrançoisFévrier，Eric Esnault和Sylvain Ducasse），Lynda-Rose Mottagan为配音，Mary Osborne-Pellegrin进行校对讲话和CYROI平台。这项工作得到了LaRéunion大学（BonusQualitéRecherche，Dispositifs incitatifs），ConseilRégionalde LaRéunion，欧盟（CPER / FEDER）和Philancia协会的资助。 ACD是LaRéunion大学（Contrat博士）德国国立教育学院获得奖学金的获得者。

将成年野生型斑马鱼（ Danio rerio ）保持在标准光周期（14/10h光/暗）和温度（28℃）条件下。所有实验均按照法国和欧洲共同体动物研究指南（86/609 / EEC和2010/63 / EU）进行，并经当地伦理委员会批准进行动物实验。 1.建立斑马鱼急性高血糖模型<ol><li>通过将400mg三卡因粉末溶解在97.9mL水和2.1mL 1M Tris / HCl缓冲液（pH9）中来制备三卡因（MS-222）的储备溶液。将pH调节至7，并将其等分储存在-20°C。 </li><li>通过将5mL三卡因（储备溶液）加入到100mL水中（最终三氯氰酸盐浓度:0.02％），为成年斑马鱼准备麻醉剂。 </li><li>将一个斑马鱼（3至6个月）从其坦克转移到麻醉剂，直至停止移动。 </li><li>回覆使用切割移液管移动鱼，并在吸收性纸巾上快速干燥。 </li><li>称重鱼</li><li>准备溶解在1X PBS中的D-葡萄糖注射器，注射50μLD-葡萄糖（2.5g / kg体重）。 注意:例如，0.6克鱼将接受50μL的3％D-葡萄糖/ PBS溶液。 </li><li>将鱼放在背部，将注射器的针头插入腹腔。 </li><li>缓慢注射D-葡萄糖/ PBS溶液，然后将鱼放回水中。 </li><li>检查鱼，直到它完全恢复。将其返回其罐，直到达到进行血糖测量所需的时间。 注意:注射后1.5小时测量血糖。 </li></ol> 2.建立斑马鱼慢性高血糖模型<ol><li>准备一个2升的清水鱼缸。 </li><li>将40g D-葡萄糖溶解在2L鱼水中，以得到最终D-葡萄糖浓度111 mM。 </li><li>在含D-葡萄糖的水中浸入5至7个成年斑马鱼。 </li><li>每2天更换D-葡萄糖鱼水，以避免细菌或其他微生物的生长。 </li><li>处理14天后，将鱼短暂置于淡水中，以便在进行血糖值测量之前除去身体外的D-葡萄糖。 </li></ol> 3.测量斑马鱼血糖水平<ol><li>用冰盖上鱼，以便快速安乐死。 注意:不要使用过量的三卡因，因为这会引起血糖水平的大幅变化。不要将鱼留在冰中太长时间，因为这会导致血液凝结。 </li><li>用吸收性纸巾擦拭鱼，以除去所有的水，并在血糖测量期间避免血液稀释。 </li><li>使用解剖镊子取下眼睛，等待眼睛充满血液。 </li><li>将测试条放在血糖仪上，将条带插入眼睛腔。 </li><li>测量血糖水平。 </li></ol> 4.分析高血糖后脑细胞增殖<ol><li> 解决方案和缓冲:要求和准备 <ol><li>通过向900mL蒸馏水（dH 2 O）中加入100毫升10倍的PBS制备1升1x PBS并混合。 </li><li>用0.2％洗涤剂（PBS-T;参见材料表）制备1x PBS。 注意:为了制备1L PBS-T，加入2mL洗涤剂（参见材料表 ）至1L PBS。 </li><li>准备乙醇系列（100％x2; 95％; 85％; 70％; 50％和30％）和0.85％NaCl。 </li><li>制备封闭缓冲液:含有0.5％至1％奶粉的PBS-T。 注意:为了制备200 mL封闭缓冲液，在200 mL PBST中加入2 g奶粉。 </li><li>准备抗原检索缓冲液，柠檬酸钠。 注意:为了制备1升柠檬酸钠缓冲液，加入2.94克柠檬酸三钠钠盐脱水至1升dH 2 0.将pH调至6，然后再填充至1 L. </li><li>制备4％多聚甲醛-PBS缓冲液，加入4g多聚甲醛至100mL的1x PBS。在58-60℃搅拌下加热，直到其完全溶解。 </li></ol></li><li> 免疫组织化学样品制备:固定和脱水 <ol><li>在测量血糖水平后，通过切割鳃后面的头部将头部与身体分开。 注意:或者，可以将大脑直接提取和冷冻用于其他实验，如mRNA提取。 </li><li>在4℃溶解于PBS（PFA-PBS）的4％多聚甲醛中将头固定过夜。 </li><li>第二天，在PBS中简单地洗头。 </li><li>用显微镜小心解剖脑部。用PBS冲洗固定头，使用针头将头部固定在解剖显微镜下。用镊子去除眼睛和头顶部。 Carefully提取大脑并将其置于1x PBS中。 </li><li>用1x PBS连续洗涤30分钟，0.85％NaCl 30分钟，70％EtOH / 0.85％NaCl（v / v）15分钟，70％EtOH 15分钟（两次），85％EtOH 20 min，95％EtOH 20分钟，100％EtOH 20分钟（两次）。在最终的100％乙醇溶液中孵育过夜。 注意:在石蜡包埋之前，脱水的脑可以在4℃的100％EtOH中保留数月。 </li></ol></li><li> 免疫组织化学样品制备:石蜡包埋的组织制备 <ol><li>将大脑放在一个小玻璃烧杯中。 注意:不要使用塑料容器，因为甲苯会损坏一些塑料。 </li><li>取出乙醇并用甲苯代替，因为大脑必须被甲苯完全回收。执行两个30分钟的甲苯浴。 </li><li>取出甲苯并将大脑置于嵌入式盒中。将封闭的盒子放在熔化的石蜡系列烧杯中58-60℃（每个石蜡烧杯中30分钟）。打开加温夹层钳。在石蜡浴的末端，取出盒子并将一些液体石蜡倒入模具中。 </li><li>将熔化的石蜡倒入模具中，将脑部放入内部。使用加温夹层钳定向大脑，使用大脑的前后方向作为指导。让石蜡在包埋机的冷却部分上硬化。 </li><li>由于技术原因，将大脑沿着前后轴定位。在解开石蜡块之后，将其固化并固定在盒子上，使熔融的石蜡以正确的方向进行横向切片。 </li><li>将石蜡块插入切片机的手臂。切割50μm厚的部分，直到达到脑样本水平。修剪石蜡块以获得一个空中飞行器，并将切片厚度调整为7μm。使用油漆刷收集石蜡带并将其放在黑色的pap上呃。每3至4节轻轻切割一次。 </li><li>将一个滑块放在暖气板上，并用dH 2 O水将其盖住。 </li><li>将切割的丝带轻轻放在水面上。当石蜡带充分展开/展开时，用吸收性纸巾去除水分。最后删除药液。从载玻片上取出水，让它们在30-40℃左右的温暖板上干燥至少3小时。 </li></ol></li><li> 免疫组化方法 <ol><li>通过将三分之一的二甲苯容器放置在每个容器中7分钟来除去石蜡。 </li><li>将载玻片放入两个100％乙醇的容器中，每次2分钟，然后加入95％EtOH，85％EtOH，70％EtOH和30％EtOH的浴，每次30秒，将部分加水。最后，简单地将幻灯片放在dH 2 O中，并在PBS中两次，每次5分钟。 </li><li>通过在微波炉中的柠檬酸盐缓冲液中孵育切片进行抗原恢复（在500W下2分钟）直到缓冲区开始沸腾。让幻灯片在室温下恢复15分钟。 </li><li>在PBST中洗涤三次，每次5分钟，并在含有1％牛奶的PBST中封闭切片45分钟。用阻断缓冲液（ 即 PBST，1％牛奶）稀释的初级抗体（ 例如增殖细胞核抗原（PCNA）用于增殖细胞染色; 1/100）孵育过夜。 </li><li>在PBST中洗涤3次，每次5分钟，并用封闭缓冲液和DAPI（1/500）稀释的合适的二次抗体（ 例如山羊抗小鼠Alexa Fluor 488; 1/200）孵育切片90分钟。 </li><li>在PBST中洗涤三次，每次5分钟，并用荧光安装介质安装载玻片（参见材料表）。 </li><li>使用落射荧光和/或共焦显微镜分析染色。 </li><li>定量至少三种不同的感兴趣区域的至少三个连续的脑切片上的脑细胞增殖imals。 注意:或者，如前所述，可以在琼脂糖中进行脑嵌入进行vibratome切片和自由浮动免疫组织化学。 </li></ol></li><li> 研究高血糖对脑修复机制的影响 注意:或者，急性和慢性高血糖症的脑修复调查可以在端脑刺伤伤后进行，如前所述， 24,25,26 。简述: <ol><li>麻醉成人斑马鱼与三叉戟。 </li><li>将鱼放在解剖显微镜下面。 </li><li>用一只手握住鱼。 </li><li>另一方面，将30 G注射器通过颅骨垂直插入右端脑半球的内侧区域。 </li><li>将鱼放回鲜鱼水，D-葡萄糖补充水（111mM），或控制鱼水。允许鱼在伤后7天生存。 注意:有关其余步骤，请参阅步骤4。 </li></ol></li></ol> 5.通过PET / CT在斑马鱼:氟脱氧葡萄糖（[18F] -FDG）中成像放射性标记的分子的生物分布来分析葡萄糖代谢<ol><li>准备一个含有20 MBq [18 F] -FDG盐水溶液的50μL注射器。 </li><li>将注射器放在辐射防护屏幕的后面。 </li><li>麻醉三叉戟的成年斑马鱼</li><li>将鱼放在防辐射屏幕的后面。 </li><li>将[18F] -FDG注入腹膜腔。用小纸巾擦拭注射部位，以防止可能从鱼肚泄漏出来的剩余[18F] -FDG。 </li><li>使用放射性同位素校准器来测量注射器和组织上包含的剩余活性，以计算精确的注射剂量。 </li><li>把鱼放在一个ew，用三叶草浸泡的小片吸收性组织轻轻地包裹起来。将吸收性组织的鱼放在PET / CT成像仪的床上。 </li><li>将床插入PET / CT成像系统并开始采集。 </li><li>继续进行PET / CT采集。 </li><li>在程序结束时，将鱼放回少量鲜鱼水中。 注意:或者，在[18F] -FDG注射后，鱼可以放回少量淡水10分钟至1小时以促进[18F] -FDG的扩散，然后麻醉鱼再次用于PET / CT成像。 </li></ol>

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(90), e51753 (2014).</li><li>Diotel, N., 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=Effects+of+estradiol+in+adult+neurogenesis+and+brain+repair+in+zebrafish.">Effects of estradiol in adult neurogenesis and brain repair in zebrafish.</a> Horm Behav. 63 (2), 193-207 (2013).</li><li>Rodriguez Viales, R., 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=The+helix-loop-helix+protein+id1+controls+stem+cell+proliferation+during+regenerative+neurogenesis+in+the+adult+zebrafish+telencephalon.">The helix-loop-helix protein id1 controls stem cell proliferation during regenerative neurogenesis in the adult zebrafish telencephalon.</a> Stem Cells. 33 (3), 892-903 (2015).</li><li>Kaslin, J., Ganz, J., Brand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation,+neurogenesis+and+regeneration+in+the+non-mammalian+vertebrate+brain.">Proliferation, neurogenesis and regeneration in the non-mammalian vertebrate brain.</a> Philos Trans R Soc Lond B Biol Sci. 363 (1489), 101-122 (2008).</li><li>Kroehne, V., Freudenreich, D., Hans, S., Kaslin, J., Brand, M. <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+adult+zebrafish+brain+from+neurogenic+radial+glia-type+progenitors.">Regeneration of the adult zebrafish brain from neurogenic radial glia-type progenitors.</a> Development. 138 (22), 4831-4841 (2011).</li><li>Alunni, A., Bally-Cuif, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+view+of+regenerative+neurogenesis+in+vertebrates.">A comparative view of regenerative neurogenesis in vertebrates.</a> Development. 143 (5), 741-753 (2016).</li><li>M&#228;rz, M., Schmidt, R., Rastegar, S., Str&#228;hle, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+response+following+stab+injury+in+the+adult+zebrafish+telencephalon.">Regenerative response following stab injury in the adult zebrafish telencephalon.</a> Dev Dyn. 240 (9), 2221-2231 (2011).</li><li>Wullimann, M., Rupp, B., Reichert, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuroanatomy of the zebrafish brain: A topological atlas. , 1-144 (1996).</li><li>Pellegrini, 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=Identification+of+aromatase-positive+radial+glial+cells+as+progenitor+cells+in+the+ventricular+layer+of+the+forebrain+in+zebrafish.">Identification of aromatase-positive radial glial cells as progenitor cells in the ventricular layer of the forebrain in zebrafish.</a> J Comp Neurol. 501 (1), 150-167 (2007).</li><li>Zupanc, G. K., Hinsch, K., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation,+migration,+neuronal+differentiation,+and+long-term+survival+of+new+cells+in+the+adult+zebrafish+brain.">Proliferation, migration, neuronal differentiation, and long-term survival of new cells in the adult zebrafish brain.</a> J Comp Neurol. 488 (3), 290-319 (2005).</li><li>Sarras, M. P., Intine, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Zebrafish+as+a+Disease+Model+Provides+a+Unique+Window+For+Understanding+the+Molecular+Basis+of+Diabetic+Metabolic+Memory.">Use of Zebrafish as a Disease Model Provides a Unique Window For Understanding the Molecular Basis of Diabetic Metabolic Memory.</a> iConcept Press. , 2611-2619 (2013).</li><li>Connaughton, V. P., Baker, C., Fonde, L., Gerardi, E., Slack, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternate+Immersion+in+an+External+Glucose+Solution+Differentially+Affects+Blood+Sugar+Values+in+Older+Versus+Younger+Zebrafish+Adults.">Alternate Immersion in an External Glucose Solution Differentially Affects Blood Sugar Values in Older Versus Younger Zebrafish Adults.</a> Zebrafish. , (2016).</li><li>Prasad, S., Sajja, R. K., Naik, P., Cucullo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetes+Mellitus+and+Blood-Brain+Barrier+Dysfunction:+An+Overview.">Diabetes Mellitus and Blood-Brain Barrier Dysfunction: An Overview.</a> J Pharmacovigil. 2 (2), 125 (2014).</li></ol>

没有披露潜在的利益冲突。

这项工作描述了在斑马鱼中建立急性和慢性高血糖模型的各种方法。这些程序的主要优点是:（1）允许减少用于研究的哺乳动物数量，（2）易于建立和快速实施，（3）它们是经济的。因此，这些模型允许调查高血糖对大量动物的影响，以研究其对不同生理过程的影响，包括动脉粥样硬化血栓形成，心血管功能障碍，视网膜病变，血脑屏障渗漏以及组成型和再生性神经发生。这项工作描述了如何进行高血糖对正常或损伤诱导条件下脑细胞增殖的影响的调查。 慢性高血糖过程的一个关键限制是，在一些实验中，一些鱼在慢性浸泡后不显示高血糖在D-葡萄糖水（111mM，14天）中。以前，Dorsemans及其同事（2016年）估计响应性和非反应性鱼类的百分比分别为83％和17％。鱼可能会根据年龄，性别和能力，通过制造更多的胰腺β细胞34,35来补偿高血糖症的个体易感性。对于急性高血糖症，注射后1.5 h血糖水平相当均匀，证明了该方法的健壮性。 该程序的关键步骤涉及血糖水平测量。在极少数情况下，填充眼腔的血液量太低，不能载入血管计测试条。此外，鱼不应该留在冰上太久以避免血液凝固。然而，它们应该保持在冰上足够的时间以确保诱导麻醉ia和动物的死亡。同样重要的是，对于急性高血糖症，注射的D-葡萄糖的体积应该改变以考虑到鱼的大小。 50μL腹腔注射液设计用于中型鱼（0.5g）。事实上，一条小鱼可能无法接受50μL的腹腔注射，并且必须减少注射体积以防止动物的痛苦，并避免溶液被压力直接推回。 另一关键步骤是成人端脑刺伤伤口的再现性;这需要一些技术经验。此外，应在感兴趣区域的三个连续部分和至少三只动物中进行计数。大脑区域的自动计数可以揭示高血糖对神经发生过程的全球影响的重要信息。 使用斑马鱼的另一个原因ish用于使用PET / CT监测放射性标记分子的生物分布的能力。在这里，[18F] -FDG被使用，其分布遍及斑马鱼身体被显示，特别包括脑和脊髓。当确定潜在治疗剂在体内模型中的递送和生物累积时，这些技术是特别有意义的。这种技术也代表了一种替代方法来研究某些分子穿过血脑屏障的能力，并确定其在生理或病理生理条件下对中枢神经系统的潜在影响。事实上，高血糖和低血糖已知可调节血脑屏障通透性36 。 在腹膜内注射后斑马鱼PET / CT成像的一个关键限制是麻醉鱼的必要性，以避免在采集过程中发生任何运动。这种麻醉可以大大降低心率，从而降低放射性示踪剂的生物分布。为了解决这个问题，根据成像方案和所用放射性同位素的半衰期，鱼可以被注入并允许在淡水中回收数分钟或数小时。此外，腹膜内注射可能导致信号在腹膜腔中的强烈积累。 总而言之，这项工作描述了建立斑马鱼高血糖模型和监测放射性标记分子分布的有效方法。这种方法可以开启一个关于调查代谢紊乱对脑体内平衡的影响以及潜在治疗剂的生物分布的研究领域。

高血糖定义为血糖过高。尽管可以反映急性压力的情况，但高血糖也是常常导致糖尿病诊断，糖尿病，胰岛素分泌和/或抵抗的慢性障碍的病症。 2016年全球糖尿病患者人数达到4.22亿，每年有150万人死于该病，成为重大的健康问题1 。事实上，不受控制的糖尿病导致影响心血管系统，肾脏和周围和中枢神经系统的几种生理学障碍。 有趣的是，急性和慢性高血糖症可能会改变认知功能，并有助于痴呆和抑郁症2,3,4,5,6。另外，病人入院高血糖与缺血性卒中7,8,9,10,11之间的功能，神经和生存结果较差有关。还显示高血糖/糖尿病影响成人神经发生，一种导致新神经元产生的过程，通过影响神经干细胞活性和神经元分化，迁移和存活2,12 。 与哺乳动物相比，硬骨鱼如斑马鱼在整个大脑中显示出强烈的神经源性活动，并且在成年期13,14,15,16期间表现出突出的脑修复能力。值得注意的是，由于neu的持续存在，这种能力是可能的ral干/祖细胞，包括放射状胶质细胞和神经母细胞17,18,19 。此外，斑马鱼近来已成为研究代谢紊乱的典范，包括肥胖和高血糖/糖尿病20,21,22 。 虽然斑马鱼是一个公认的高血糖和神经发生模型，但很少有研究调查了高血糖对脑内稳态和认知功能的影响12,23。为了确定高血糖对组成型和损伤诱导的脑细胞增殖的影响，通过腹膜内注射D-葡萄糖产生急性高血糖模型。此外，通过将鱼浸入补充水的水中再现慢性高血糖模型D-葡萄糖12 。斑马鱼在研究中表现出很多优势。它们在开发的第一阶段便宜，易于提高和透明，并且其基因组已被测序。在这项工作的背景下，他们还展示了几个额外的优点:（1）他们与人类共享类似的生理过程，使其成为生物医学研究的关键工具; （2）鉴于其广泛而强的神经源性活性，它们可以快速调查高血糖对脑内稳态和神经发生的影响;和（3）它们是替代模型，允许减少研究中使用的哺乳动物的数量。最后，斑马鱼可用作测试放射性标记分子和潜在治疗剂使用PET / CT的生物分布的模型。 以下程序的总体目标是视觉记录如何建立斑马鱼急性和慢性高血糖模型，使用zeb在高血糖条件下评估脑重塑，并使用PET / CT监测放射性标记的分子（这里为[ 18 F] -FDG）。

<table border="0" cellpadding="0" cellspacing="0" width="738">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:328px;">1mL Luer-Lok Syringe</td>
			<td style="width:151px;">BD, USA</td>
			<td style="width:93px;">309628</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">4&#39;,6&#39;-diamidino-2-phenylindole (DAPI)</td>
			<td>Sigma-Aldrich, Germany</td>
			<td style="width:93px;">D8417</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">7 mL bijou container plain lab</td>
			<td style="width:151px;">Dutscher, France</td>
			<td style="width:93px;">080171</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">D-glucose</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>67021</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Digital camera</td>
			<td>Life Sciences, Japan</td>
			<td>Hamamatsu ORCA-ER</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Disposable base molds&#160;</td>
			<td>Simport, Canada</td>
			<td>M475-2</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Donkey anti-rabbit Alexa fluor 488</td>
			<td>Life Technologies, USA</td>
			<td>A21206</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Embedding center</td>
			<td>Thermo Scientific, USA</td>
			<td>Shandon Histocentre 3</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fluorescence microscope</td>
			<td>Nikon, Japan</td>
			<td>Eclipse 80i</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorodeoxyglucose (18F-FDG)</td>
			<td>Cyclotron, France</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Glucometer test strip</td>
			<td>LifeScan, France</td>
			<td>One-Touch 143 Ultra</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Goat anti-mouse Alexa fluor 594</td>
			<td>Life Technologies, USA</td>
			<td>A11005</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">In-Vivo Imaging System</td>
			<td>TriFoil Imaging, Canada</td>
			<td>Triumph Trimodality&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Microtome</td>
			<td>Thermo Scientific, USA</td>
			<td>Microm HM 355 S</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Monoclonal mouse anti-PCNA</td>
			<td>DAKO, USA</td>
			<td>clone PC10</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>P6148-500G</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Polyclonal rabbit anti-GFAP</td>
			<td>DAKO, USA</td>
			<td>Z033429</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Slide drying bench</td>
			<td>Electrothermal, USA</td>
			<td>MH6616</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Sodium chloride</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Sodium citrate trisodium salt dehydrate&#160;</td>
			<td>Prolabo, France</td>
			<td>27833.294</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Sterile needle</td>
			<td>BD Microlance 3</td>
			<td>30 G 1/2 ; 0.3 mm x 13 mm</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Student Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>91150-20</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Student surgical scissors</td>
			<td>Fine Science Tools</td>
			<td>91400-14</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Superfros Plus Gold Slides</td>
			<td>Thermo Scientific, USA</td>
			<td>FT4981GLPLUS</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Surgical microscope</td>
			<td>Leica, France</td>
			<td>M320-F12</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tissue embedding cassettes</td>
			<td>Simport, Canada</td>
			<td>M490-10</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tissue embedding medium</td>
			<td>LeicaBiosystems, USA</td>
			<td>39602004</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Toluene</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>244511</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:328px;">Tricaine MS-222</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>A5040</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Triton X100</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>X100-500 mL</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Vectashield medium&#160;</td>
			<td>Vector Laboratories, USA</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Xylene</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>534056</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fish Strain</td>
			<td>AB</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Saline phosphate buffer (10X PBS) pH 7.4 (for 1 liter)</td>
			<td></td>
			<td></td>
			<td>For preparing 10X PBS, add the following&#160; salts and complete to 1 liter with distilled water</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Potassium chloride (MM : 74.55 g/mol): 2.00 g</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>746436</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Potassium phosphate monobasic (MM: 136,09 g/mol): 2.40g</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>795488</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Sodium chloride (MM : 58.44 g/mol): 80.00 g&#160;</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Sodium phosphate dibasic (MM: 141,96 g): 14,40 g</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>795410</td>
			<td></td>
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acute and chronic models of hyperglycemia in zebrafish: a method to assess the impact of hyperglycemia on neurogenesis and the biodistribution of radiolabeled molecules

高血糖是导致心血管和脑功能障碍的主要健康问题。例如，它与中风后增加的神经系统问题相关，并且显示损害神经源性过程。有趣的是，成年斑马鱼已经成为模拟高血糖/糖尿病并调查组成型和再生神经发生的相关和有用的模型。这项工作提供了开发高血糖斑马鱼模型的方法，以探讨在稳态和脑修复条件下高血糖对脑细胞增殖的影响。使用腹膜内注射D-葡萄糖（2.5g / kg体重）到成年斑马鱼中建立急性高血糖。通过将成人斑马鱼浸入含有水的D-葡萄糖（111mM）中14天来诱导慢性高血糖。对于这些不同的方法描述血糖水平测量。调查高血糖对组成型a的影响的方法通过描述端脑的机械损伤，解剖脑，石蜡包埋和切片切片，并进行免疫组织化学程序，进行再生神经发生。最后，还描述了使用斑马鱼作为研究放射性标记分子的生物分布的相关模型的方法（这里，[ 18 F] -FDG）使用PET / CT。

使用本文所述的方法，在成年斑马鱼上进行腹膜内注射D-葡萄糖（2.5g / kg体重），并且在注射后1.5小时导致血糖水平的显着增加（ 图1A ）。注射后24小时，D-葡萄糖和PBS注射的鱼12之间的血糖水平相似。对于慢性治疗，将斑马鱼浸入D-葡萄糖水（111mM）中，并在其14天治疗结束时变成高血糖（ 图1B ），如前所述12,22 。 为了研究高血糖对脑细胞增殖的影响，在诱导急性和慢性高血糖症后，对斑马鱼脑进行PCNA免疫组化。虽然急性hypergl<html> ycemia没有影响脑细胞增殖12 ，慢性高血糖症导致心室神经干细胞增殖显着降低，如Dorsemans及其同事（2016）所示。实际上，PCNA阳性细胞的数量在小疱（Vv / Vd），大脑膜（Dm）以及尾部下丘脑（LR / PR）的外侧和后部凹陷周围的区域减少（ 图2 ）。 在急性和慢性高血糖症患者机械性脑损伤后，也研究了损伤诱导的神经发生。如之前在斑马鱼脑损伤中描述的那样，发生小胶质细胞和少突胶质细胞的第一实质增殖，其次是在损伤后7天在心室层上强烈的上调增殖25,27 ，<html>急性高血糖并未调节脑实质增殖的初始步骤，相比之下，慢性高血糖症损伤后7天，脑周围脑室细胞增殖受损（ 图3 ）。 斑马鱼模型对于使用PET / CT成像监测放射性标记分子的生物分布也很有意义。在这里，将[18F] -FDG腹膜内注射入成年斑马鱼。 30分钟后，PET / CT采集显示，葡萄糖不仅分布在注射部位，而且还分布在包括大脑在内的脊髓和脊髓的头部（ 图4 ）。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55203/55203fig1.jpg" /> 图1 :斑马鱼高血糖症的急性和慢性模型 （ A ）腹膜内注射D-葡萄糖（2.5g / kg体重）导致注射后1.5小时血糖水平显着升高（n = 3） ）（ B ）将斑马鱼在D-葡萄糖水（111mM）中浸泡14天导致血糖水平显着升高（n = 15）， <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55203/55203fig1large.jpg" target="_blank">请点击此处查看该图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55203/55203fig2.jpg" /> 图2 :慢性高血糖症治疗14天后会损害脑细胞增殖。增殖细胞用PCNA抗体以绿色标记。细胞核用DAPI（蓝色）复染色。慢性的yperglycemia在小脑（ A ），大脑（ B ）以及脑室外侧和后侧凹陷周围的尾部下丘脑（ C ）中治疗14天后降低脑细胞增殖。刻度棒=120μm（A和B），200μm（C）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55203/55203fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/55203/55203fig3.jpg" /> 图3 :端脑刺伤伤口损伤后7天上调脑增殖。 （ A ）上方矢状面所示水平的斑马鱼端脑横断面示意图。模式已从斑马鱼脑图集31获取 。那里<html> d点表示增殖细胞32,33 。针表示病变部位。 （ B ）脑损伤后7天PCNA（绿色）免疫组织化学显示受损脑端脑中脑室内增殖的强烈上调。刻度棒= 200μm。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55203/55203fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" class="xfigimg" src="/files/ftp_upload/55203/55203fig4.jpg" /> 图4 :腹膜内注射后30分钟[18F] -FDG（注射20MBq）的PET / CT成像。 PET / CT成像的代表性图像显示斑马鱼身体中[18F] -FDG的广泛分布，包括头部，脑部和脊髓。files / ftp_upload / 55203 / 55203fig4large.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。

Watch this Scientific Journal Video about Acute and Chronic Models of Hyperglycemia in Zebrafish: A Method to Assess the Impact of Hyperglycemia on Neurogenesis and the Biodistribution of Radiolabeled

Acute and Chronic Models of Hyperglycemia in Zebrafish: A Method to Assess the Impact of Hyperglycemia on Neurogenesis and the Biodistribution of Radiolabeled Molecules

这项工作描述了在斑马鱼中建立急性和慢性高血糖模型的方法。目的是调查高血糖对生理过程的影响，如组成型和损伤诱导的神经发生。这项工作还突出了使用斑马鱼跟踪放射性标记的分子（这里，[ 18 F] -FDG）使用PET / CT。

斑马鱼高血糖的急性和慢性模型:评估高血糖对神经发生的影响和放射性标记的分子的生物分布的方法

Hyperglycemia is a major health issue that leads to cardiovascular and cerebral dysfunction. For instance, it is associated with increased neurological ...

acute-chronic-models-hyperglycemia-zebrafish-method-to-assess-impact

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

13.3K 次观看.  Université de La Réunion, INSERM. 这项工作描述了在斑马鱼中建立急性和慢性高血糖模型的方法。目的是调查高血糖对生理过程的影响，如组成型和损伤诱导的神经发生。这项工作还突出了使用斑马鱼跟踪放射性标记的分子（这里，[ 18 F] -FDG）使用PET / CT。 

视频: Acute and Chronic Models of Hyperglycemia in Zebrafish: A Method to Assess the Impact of Hyperglycemia on Neurogenesis and the Biodistribution of Radiolabeled Molecules

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

多能干细胞的海藻酸钠封装使用同轴喷嘴

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

科研

发育生物学

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Using Touch-evoked Response and Locomotion Assays to Assess Muscle Performance and Function in Zebrafish

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many myopathies affecting skeletal muscle function can be quickly and easily assessed in zebrafish over the first few days of embryogenesis. By 24 hr post-fertilization (hpf), wildtype zebrafish spontaneously contract their tail muscles and by 48 hpf, zebrafish exhibit controlled swimming behaviors. Reduction in the frequency of, or other alterations in, these movements may indicate a skeletal muscle dysfunction. To analyze swimming behavior and assess muscle performance in early zebrafish development, we utilize both touch-evoked escape response and locomotion assays.
Touch-evoked escape response assays can be used to assess muscle performance during short burst movements resulting from contraction of fast-twitch muscle fibers. In response to an external stimulus, which in this case is a tap on the head, wildtype zebrafish at 2 days post-fertilization (dpf) typically exhibit a powerful burst swim, accompanied by sharp turns. Our method quantifies skeletal muscle function by measuring the maximum acceleration during a burst swimming motion, the acceleration being directly proportional to the force produced by muscle contraction.
In contrast, locomotion assays during early zebrafish larval development are used to assess muscle performance during sustained periods of muscle activity. Using a tracking system to monitor swimming behavior, we obtain an automated calculation of the frequency of activity and distance in 6-day old zebrafish, reflective of their skeletal muscle function. Measurements of swimming performance are valuable for phenotypic assessment of disease models and high-throughput screening of mutations or chemical treatments affecting skeletal muscle function.

Zebrafish are an excellent model to study muscle function and disease. During early embryogenesis zebrafish begin regular muscle contractions producing rhythmic swimming behavior, which is altered when the muscle is disrupted. Here we describe a touch-evoked response and locomotion assay to examine swimming performance as a measure of muscle function.

采用触摸诱发反应和运动测定以评估在斑马鱼肌肉性能和功能

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many ...

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize mammalian adaptive immunity. Drosophila and mammalian hematopoiesis occur in spatially and temporally distinct phases to produce several blood cell lineages. Both systems maintain reservoirs of blood cell progenitors with which to expand or replace mature lineages. The hematopoietic system allows Drosophila and mammals to respond to and to adapt to immune challenges. Importantly, the transcriptional regulators and signaling pathways that control the generation, maintenance, and function of the hematopoietic system are conserved from flies to mammals. These similarities allow Drosophila to be used to genetically model hematopoietic development and disease.
Here we detail assays to examine the hematopoietic system of Drosophila larvae. In particular, we outline methods to measure blood cell numbers and concentration, visualize a specific mature lineage in vivo, and perform immunohistochemistry on blood cells in circulation and in the hematopoietic organ. These assays can reveal changes in gene expression and cellular processes including signaling, survival, proliferation, and differentiation and can be used to investigate a variety of questions concerning hematopoiesis. Combined with the genetic tools available in Drosophila, these assays can be used to evaluate the hematopoietic system upon defined genetic alterations. While not specifically outlined here, these assays can also be used to examine the effect of environmental alterations, such as infection or diet, on the hematopoietic system.

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells.

方法来检查淋巴腺和血细胞中<em&gt;果蝇</em&gt;幼虫

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

Building Finite Element Models to Investigate Zebrafish Jaw Biomechanics

Skeletal morphogenesis occurs through tightly regulated cell behaviors during development; many cell types alter their behavior in response to mechanical strain. Skeletal joints are subjected to dynamic mechanical loading. Finite element analysis (FEA) is a computational method, frequently used in engineering that can predict how a material or structure will respond to mechanical input. By dividing a whole system (in this case the zebrafish jaw skeleton) into a mesh of smaller 'finite elements', FEA can be used to calculate the mechanical response of the structure to external loads. The results can be visualized in many ways including as a 'heat map' showing the position of maximum and minimum principal strains (a positive principal strain indicates tension while a negative indicates compression. The maximum and minimum refer the largest and smallest strain). These can be used to identify which regions of the jaw and therefore which cells are likely to be under particularly high tensional or compressional loads during jaw movement and can therefore be used to identify relationships between mechanical strain and cell behavior. This protocol describes the steps to generate Finite Element models from confocal image data on the musculoskeletal system, using the zebrafish lower jaw as a practical example. The protocol leads the reader through a series of steps: 1) staining of the musculoskeletal components, 2) imaging the musculoskeletal components, 3) building a 3 dimensional (3D) surface, 4) generating a mesh of Finite Elements, 5) solving the FEA and finally 6) validating the results by comparison to real displacements seen in movements of the fish jaw.

Finite Element Analysis is a frequently used tool to investigate the mechanical performance of structures under load. Here we apply its use to modeling the biomechanics of the zebrafish jaw.

建立有限元模型，研究斑马鱼下颌生物力学

Skeletal morphogenesis occurs through tightly regulated cell behaviors during development; many cell types alter their behavior in response to mechanical ...

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 ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

成人斑马鱼损伤模型研究泼尼松对再生骨组织的影响

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

HUVEC Tube-formation Assay to Evaluate the Impact of Natural Products on Angiogenesis

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the formation of new blood vessels and involve several signal transduction pathways. Here we show that the tube formation assay is a simple in vitro method to evaluate the impact of natural products on angiogenesis and to investigate the molecular mechanisms involved. In particular, in the presence of the water extract of Ruta graveolens (RGWE), endothelial cells are no longer able to form a cell-cell network and that the RGWE effects on human umbilical vein endothelial cell (HUVEC) tube formation is abolished by the constitutive activation of MEK.

Here, we evaluate the effects of the water extract of Ruta graveolens on vessel network formation by using a tube formation assay on a gelled basement matrix.

HUVEC 管形成测定评估天然产物对血管生成的影响

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the ...