笔者想感谢实验室成员斑马鱼的保养和维护。这项工作是由美国国立卫生研究院，美国国家研究服务奖A155397到EA哈维和NIH R01GM074827安娜Huttenlocher支持。

成人和胚胎斑马鱼维持在按照威斯康星大学麦迪逊分校的研究动物资源中心的大学。 1.准备显微注射针<ol><li>在拉5，空气时开始的空气压力200，热量502，拉90，速度80，时间70，广播时间:准备使用微量牵引装置具有以下设置的薄壁玻璃毛细管注射针晶（1.0 OD / 0.75的ID）在拉5月底。 </li><li>使用细镊子，断绝拉到针的尖端，以使顶端开口的直径为大约10微米。 </li></ol> 2.准备幼虫注射菜<ol><li>冲洗凝胶梳子约为4-5毫米的无菌水车道宽度并晾干。 </li><li>准备在E3的介质19和微波炉的1.5-2％的高熔体琼脂糖溶液，直至溶液澄清。一旦冷却，倒入一些琼脂糖到培养皿（100×15毫米）和漩涡，加入足够的琼脂糖完全覆盖培养皿底部。 </li><li>一旦琼脂糖层已经凝固，将漂洗并干燥凝胶梳子上顶，以使非精梳端只是搁置在培养皿的顶部和梳理端正在触摸琼脂糖。确保梳子是尽可能的水平，创造了30°角相对于所述底部琼脂糖层。 </li><li>倾额外少量琼脂糖在梳子和底部琼脂糖层之间的界面，以使新鲜的琼脂糖层覆盖所述梳状的孔中。允许取出梳子之前完全冷却。用枪头，从水井中删除任何悬垂件琼脂糖。 </li><li>倒在4℃下在注塑模具及商店的顶部E3介质。 </li><li>每次使用前，更换新鲜培养基和温暖注入坡道在28.5°C，至少注射前一小时。 </li><li>立即注射之前，取代E3上的注射用斜含有200微克/ ml的3-氨基苯甲酸甲酯（三卡因）的E3介质。 </li></ol> 3.准备S. iniae接种<ol><li>准备并高压灭菌补充有0.2％酵母提取物和2％蛋白胨（THY + P）托德Hewitt肉汤培养基:30g / L的托德休伊特，2克/升酵母提取物，将20g / L的蛋白胨。对琼脂平板，加14克/ L琼脂。 </li><li>夜感染吹打冷冻细菌库存100微升等分试样到10ml，THY + P肉汤在密封的15ml试管之前准备的细菌培养物。孵育过夜不用搅拌，在37℃。经过14-16小时的增长，使用过夜培养既可以使冷冻股票或在注射使用做准备。 </li><li>通过将1毫升在500μl80％的甘油的过夜培养物在1.7毫升离心管使冷冻储存。为了避免反复冻融，从该混合并储存在-80℃下使100μl的一次性使用的等分试样。 </li><li>对于S. iniae用于infecti通过加入0.1过夜培养毫升100 10毫升培养的总体积为9.9，TH​​Y + P肉汤毫升:项，稀释过夜培养1。成长在37°C时不搅拌约4-5小时。监测使用NanoDrop分光光度计的光密度（OD）在600nm和收获细菌在中期对数生长期时，OD 600 nm的到达0.250-0.500。为0.250的OD 600毫微米相当于约10 8个菌落形成单位（CFU）/毫升。 </li><li>沉淀将1ml细菌培养物在1.7毫升离心管中，在1500×g离心5分钟。重悬在1ml新鲜的PBS并重复。测量细菌在PBS的OD 600纳米，沉淀，重悬在PBS中以达到所需的浓度。 </li><li>为了帮助在显微注射的可视化，事先添加酚红对细菌悬浮液以注射为0.1％的最终浓度。 </li><li>对于涉及在注射热灭活细菌的实验中，加热该细菌在PBS中在95℃下进行30分钟。确认热杀死过程由固体，T​​HY + P琼脂板上电镀的注射体积（大约1 NL）中并在37℃温育过夜减压活菌到检测不到的水平的数目。 </li></ol> 4.标签S. ËINIA用CellTracker红色荧光染料<ol><li>标记活细菌细胞，制备CellTracker荧光染料或等效的储液。作为所使用的染料来在20×50微克等份粉末，需要在DMSO中进行重新悬浮，微升DMSO中添加7.3到管以得到10mM的储备浓度。 </li><li>测试一系列浓度的染料（ 例如，0.5-25μM）上的细菌，以确定污渍的细胞的最低最佳浓度。快速分裂的细胞和更长的实验可能需要染料的浓度较高。 <ol><li>上述（第3节）所描述的颗粒1.0毫升细菌培养在1.7毫升管离心。悬浮在1球毫升新鲜的PBS，并添加染料的适当体积的细菌培养物。 </li><li>孵育不搅拌，在37℃下进行30分钟。降速的细菌重悬在1ml预热THY + P肉汤孵育不搅拌另外30分钟，在37℃。 </li><li>降速的细菌和测量OD600纳米及以上的（第3节）详见稀释细菌注射前，在PBS洗两次。我们通常注入约100 CFU 1 NL注射量为白细胞招聘和吞噬的研究。 </li></ol></li></ol> 5.准备斑马鱼幼虫的感染<ol><li>之前设置了繁殖对夜间和所描述的罗森等27孵育胚胎在E3中在28.5°C，直到准备感染收集胚胎。 </li><li>对于斑马鱼，将被成像，防止颜料（黑化）的开发中加入N-二苯基硫脲（PTU）的E3的培养基中于24小时受精后（HPF）为0.2纳米的最终浓度。 </li><li>对于涉及胚胎感染2岁旦，dechorionate胚胎一双细镊子手动。可替代地，dechorionate胚胎通过去除E3和用链霉蛋白酶（2毫克/毫升）约5分钟，或直至轻柔吹打替换破坏的胚胎从绒毛膜。大部分幼虫自然应该由3 DPF孵化。 </li><li>通过将dechorionated幼虫到含有200微克/毫升三卡因E3介质麻醉斑马鱼几分钟之前感染。 </li></ol> 6.耳泡注射液S. iniae进入第三天高龄幼虫<ol><li>打开微量，并设定​​时间范围为"毫秒"。打开对二氧化碳罐阀让气体进入线。该微量的压力应为大约20 PSI。由黑色圆形旋钮为增量的顺时针转动调节在微量单元中的压力缓解压力或逆时针旋转的下降压力。 </li><li>涡准备S.在酚红和PBS，并使用微加载提示iniae文化装载2-3微升培养成一把拉住毛细管注射针头。 </li><li>安装在连接到立体显微镜下一个磁性支架并将其定位显微加载针，使针是在约一45-65°的角度相对于所述显微镜的基极。 </li><li>按微量的脚踏上以分配一滴接种到针的尖端。测量使用在显微镜的目镜比例尺下降的直径。 <ol><li>可替代地，通过注射体积为矿物油对带有标尺条的玻璃显微镜载玻片一滴估算滴的直径。液滴的直径应为约0.10mm，大约是一个1 NL体积。 </li><li>通过调整时间设定在调整墨滴大小的微量或通过剪切掉更多针尖细镊子。需要注意的是喷射时间应在20 - 35毫秒，以避免造成过多的组织损伤。 </li></ol></li><li>使用塑料移液管，移12麻醉幼虫放入注射模具的每个孔中。 </li><li>使用玻璃棒，毛发循环，或塑料尖端轻轻地定位幼虫，使磁头指向朝向显微镜和卵黄囊的背面是对井的左侧。点朝天花板幼虫了左耳。 </li><li>期待通过立体显微镜的目镜中，使用在显微旋钮排队装入针与耳泡，使得两者都在同一视场。皮尔斯的耳泡与针尖的外皮层，以使针尖仅仅是囊泡内部。 </li><li>按下脚踏上注入1 NL S的所需剂量的iniae。请务必使用足够低的压力，以便不破裂空腔。如果注射是成功的，耳泡，但不是周围组织，应填写与酚红接种（ 图1艾 ）。立即从注射板删除任何错误注入鱼。 </li><li>仔细缩回针从幼虫和用手移动喷射板，以使下一个幼虫是图。这样做在放大5倍。 注意:在针的回缩可能导致耳泡的外一些细菌，其可以歪斜存活和白细胞募集的结果的沉积。为了避免这种情况，建议注入荧光色素标记的细菌和视觉扫描荧光显微镜下注射的幼虫以除去其中这已发生的任何幼虫。一个正确的注射幼虫所示（ 图1碧 ）。 </li><li>为了确保注射体积/接种物保持不变在实验的过程中，注入一滴细菌的Suspension到含有100微升无菌PBS的每48 个胚胎后1.7毫升离心管中。板中的100μl的上，THY + P琼脂平板在37℃下过夜，以确定在注射体积的CFU。 </li><li>当12幼虫在注射坡道的整个集团已经注入，小心使用塑料移液管从井中取出幼虫，并放置到一个新的培养皿（35×10 平方毫米）。取下三卡因溶液，并用约新鲜E3媒体2毫升取代，让幼虫恢复。 </li><li>吸管注入幼虫成单个孔96孔板的孵育在28.5℃。监测幼虫随着时间的推移生存在这些板块或更高版本的成像或CFU计数删除。 </li></ol>中性粒细胞7.苏丹黑染色注意:下面的步骤可以在室温下在定轨振荡器，除非OT进行在小培养皿（35×10mm的2）herwise说明。对于每个步骤，试剂的使用量是约每皿2毫升，使用刚好足够的液体，以完全覆盖幼虫。 <ol><li>对受感染的幼虫的​​固定，使用玻璃巴斯德吸管除去E3介质，并替换为在PBS溶液中的冰冷却的4％多聚甲醛。立即将鱼在4℃安乐死。修复一夜之间在4℃。 </li><li>在PBS，每次5分钟洗涤3次。 </li><li>染色用苏丹黑（0.18％库存1:5稀释在70％的乙醇，0.1％苯酚）处理30分钟至5小时。使用立体显微镜来检查，如果中性粒细胞颗粒扛起了污点。 </li><li>执行一系列的5分钟洗涤开始在70％乙醇中的单次洗涤之后的1的单一洗涤:1的比例70％乙醇和PBS，随后在PBS中进行最后的清洗。 </li><li>再水合到PBS加0.1％吐温（PBT）。 </li><li>以清除颜料从幼虫，洗净，以1％的氢氧化钾/ 1％的过氧化氢为约6-10分钟。监测样本closely和大约5分钟后，检查在立体显微镜下的幼虫，看看颜料多已被清除。如果溶液被留在过长，卵黄囊会膨胀和爆裂。 </li><li>在PBS中洗涤3次，每次5分钟。 </li><li>储存在PBS或PBT样品在4℃下长达1周，直到准备图像和计数。 </li><li>图像苏丹黑染色幼虫，鱼转移到PBT，然后到80％的甘油，并放置到一个单腔抑郁症的幻灯片。位置固定下使用移液管尖端的立体显微镜幼虫。图像使用彩色数码相机立体显微镜（ 图1 AII-IV）。 </li></ol> 8.枚举从受感染的幼虫活菌<ol><li>安乐死幼虫在所需时间感染后在E3的介质冰冷200-300毫克/升三卡因溶液。 </li><li>吸管安乐死幼虫进入1.7毫升离心管。除去三卡因溶液和替换用100μl的0.2％TRITON X-100的PBS（PBSTx）。 </li><li>通过一个27G的针头上下传球10次均质幼虫。 </li><li>准备在无菌PBS匀浆的系列稀释。例如，如果感染剂量为100 CFU，吸管100微升匀浆入900微升无菌PBS中。使用新的枪头，转移100微升稀释的匀浆到900微升无菌PBS。 </li><li>板100微升哥伦比亚CNA琼脂为革兰氏阳性菌的选择性分离的稀释液，孵育在37℃下48小时。此介质将提供更大的选择比，THY + P琼脂板上，这将同时支持革兰氏阴性和革兰氏阳性细菌的生长。 </li><li>在平板上少于500单个菌落，计数菌落数并乘以稀释因子相乘以确定CFU数量。 </li></ol> 9.固定幼虫成像<ol><li>修正的幼虫在PBS溶液作为去冰 - 冷的4％多聚甲醛划在第7。 </li><li>在室温下，在成像前洗3次，每次5分钟的PBS。 </li></ol> 10.准备幼虫的实时成像<ol><li>制备1.5％低融点琼脂糖中的E3通过加热溶液在微波中，直至溶液澄清。 </li><li>将在55℃水浴琼脂糖溶液，使其冷却，但不会变硬。 </li><li>添加三卡因到琼脂糖的0.016％的最终浓度。 </li><li>用塑料移液管，放置4-5麻醉幼虫在一个立体的舞台玻璃底菜。 </li><li>除去从55℃水浴中的三卡因和琼脂糖溶液，让它冷却，在室温下搅拌1-2分钟。而琼脂糖冷却，从被麻醉的幼虫尽可能除去尽可能多的液体。 </li><li>倒入冷却的琼脂糖放入盘中，直到大约一半的表面被水覆盖。摇动盘传播的琼脂糖。需要注意的是，如果琼脂糖太热，它会杀死幼虫。此外，如果过多琼脂糖加入到皿中，在显微镜的物镜可以通过琼脂糖聚焦时击中培养皿的底部。 </li><li>用移液管，拿起那些漂浮在盘子的两侧的幼虫和吸管它们放回中心。 </li><li>根据体视显微镜，根据需要用吹风环路，长枪头或玻璃棒轻轻放置幼虫。用于耳泡的成像，定位幼虫，使得左耳泡是平贴在培养皿的底部。 </li><li>让琼脂糖冷却约10分钟，移动盘之前。幼虫可移仓，如果琼脂糖并不牢固。轻轻吸管一些三卡因和E3溶液到琼脂糖层的顶部，以保持湿润。 </li></ol>感染11.共聚焦成像<ol><li>放置玻璃底皿幼虫到倒置显微镜具有FV-1000激光扫描confoca的阶段升系统。 </li><li>针孔设定为200-300微米，使用数值孔径0.75 / 20X物镜，设置Z轴堆叠具有3-6微米的切片。 </li><li>使用连续扫描线来调整激光功率和检测器的增益为每个通道。 </li><li>使用顺序逐行扫描每个荧光通道（ 例如，488和543纳米）和微分干涉对比（DIC），进行左侧耳泡的时间间隔电影每3分钟2-6小时，观察初始招聘和吞噬中性粒细胞和巨噬细胞。对于较长的时间进程，放置在培养皿的盖以防止蒸发和干燥从琼脂糖。 </li><li>收购仍是固定的（ 图1B）的图像或生活（ 图2），在幼虫或20X 40X的放大倍率。 </li></ol> 12.光转化Dendra2标记的白细胞在耳泡注:Dendra2可以从绿色将光转换为红色荧光通过聚焦405nm激光（50-70％的激光功率应足以）上的感兴趣区域（ROI）的区域，持续1分钟。下面是一步一步协议用于FV-1000激光扫描共聚焦系统: <ol><li>使用Z堆栈扫描与488 nm和543 nm激光可视化的样本。使用连续扫描线来调整激光功率和检测器的增益。检查以确保没有不小心光转换红色荧光。 </li><li>在"图像采集控制"窗口，"刺激设置"下，选中"使用扫描仪"工具，然后选择"主"。选择405 nm激光，并定于70％的电力。使用循环选项，定义投资回报率在耳泡。 </li><li>在"刺激开始设置"中选择1帧的预激活和60000毫秒的激活时间"激活串联"。就在"获取设置"窗口中的"时间扫描标题"，选择2的0时01分00秒（一个用于前和一个用于光转化后）tervals。 注意:时间经过一系列扫描完成后，将Dendra2应已光转换到其红色荧光的状态。 </li><li>通过使用488纳米和543纳米激光器的可视化的光转换的红色荧光以及任何剩余的绿色荧光（ 图3）的Z堆叠扫描样品。 </li></ol>

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The authors declare that they have no competing financial interests.

此处所用的感染的方法是用于在2-3旦胚胎和幼虫的初始局部感染宿主的免疫应答的研究是有用的。炎症刺激，如感染，在一个封闭的腔体的焦点，如耳泡允许对嗜中性粒细胞和巨噬细胞趋化及吞噬的研究。注入细菌进入耳泡的一个需要注意的是中性粒细胞的能力，以有效地吞噬细菌在填充流体的腔可以依赖于特定的微生物。尽管大肠杆菌和枯草芽孢杆菌 ，不容易通过在耳泡28嗜中性粒细胞吞噬，我们已经发现，嗜中性粒细胞能够吞噬既绿脓杆菌和S. iniae在此位置16，24。局部感染也很有研究的各种病原体侵袭的时候。为了引起全身性感染以下我njection成一个封闭的空腔，如耳泡，微生物必须能够遍历物理主机的障碍。可替代地，不同的病原体可能依赖于宿主细胞的运输和传播感染的初始位点。这使得局部注射的改变比较毒力株有用。 期间显微注射，以避免细菌沉降和堵塞针，改变针或者之后的每个条件或之后约50幼虫。这将有助于确保该悬浮液在针是更均匀的。如果在针沉降细菌似乎是一个问题，PBS中混合，2％PVP40，和10％的甘油可以帮助保持一个均匀的悬浮液。以确保每个幼虫被注入了细菌的大致相同的编号，检查CFU计数通过立即均质幼虫注射后及电镀CNA琼脂匀浆。 CNA琼脂将用于培养的革兰氏阳性细菌的生长进行选择，不仅S. iniae，但它会提供的注射的剂量如何一致是每个单独的幼虫之间的想法。以每100幼虫CFU靶接种物，通常有75-150之间的一个CNA板菌落。非S的数iniae革兰氏阳性克隆在CNA板上生长可能是非常低的，因为大多数的PBS注射的鱼通常会导致0-20殖民地之间。在感染期间后的时间点，这种情况并不少见，要有高达变异半个日志中的幼虫感染同一感染剂量之间菌落计数。这可能代表个别幼虫之间的细微差别在其控制感染能力或可能反映在幼虫或E3介质中培养的革兰氏阳性菌的量的差异。 而注入的耳泡，它不注入过大的体积或具有过高的压力，因为这可能会导致模腔破裂是很重要的。进样体积笑ULD保持约1 NL。如果针太大，显微注射可能损坏周围的组织，这可能会影响宿主的白细胞的募集至感染部位，或者可以使细菌漏出耳泡的。同样重要的是，以确认注射到正确的空间。如果针被插入过深，则可能捅通过耳囊或者它可撞击围绕耳泡流动的血管。这可能会导致细菌的沉积到血流中或囊泡外面，这可能会改变宿主反应。 另外，也可以是，当所述针被提取，一些细菌可能会被意外耳囊外沉积如图Colucci的居永等人 28，但是我们发现这仅是在注射的不足5％的情况。试图注入耳泡的中心可能有助于避免出现这种情况。在一个成功的感染，耳泡应填写瓦特第i个酚红染料，但染料不应泄漏到周围组织中。任何错误注入幼虫应立即丢弃。注射标记的细菌是确认成功注射（ 图1B）的有用方法。一种使用CellTracker染料的缺点之一是，该染料将成为稀的细菌分裂，最终防止细菌从下荧光被可视化。我们选择了红色染料，因为我们用绿色标记的中性粒细胞，并为广大研究的巨噬细胞的转基因株系。 Dendra2是octocoral Dendronephthya SP派生的photoconvertible蛋白质。它可以从一个绿色被光转换成红色荧光状态有405nm激光29。这光转化是一种非侵入性的方式来标记细胞，并跟踪他们的命运在感染过程。白细胞募集到感染部位，可光转换并随后随时间监测其C在整个宿主组织issemination（ 图3）。可替代地，白细胞可以在感染前光转换，然后监测后显微注射，以确定细胞招募到感染部位的原点。标记细菌和免疫细胞允许在体内白细胞-病原体相互作用包括招募和吞噬（ 图1B和图2）的研究。区分红色标记S. iniae从光转换白细胞的红色荧光是困难的，所以不同的荧光CellTracker染料也可以使用。这将是特别有用的，以确定该光转换免疫细胞离开耳泡的含有S. iniae。 耳囊感染方法的未来的应用可以包括测量速度和方向由嗜中性粒细胞和巨噬细胞移动响应于各种感染。感染ķinetics也可以研究以查看哪些细胞类型是第一个到达感染部位和哪些细胞类型是最稳健招募除了其中的细胞来源于或者它们传播30。除了研究招募的初始局部感染，这种感染模型可以用于最初的感染过程中，研究宿主的免疫系统的组件的功能。反义吗啉代寡核苷酸针对特定的RNA可用于击倒宿主的免疫组件，包括Toll样受体，细胞因子受体和白细胞，和表达CRISPR-CAS或TALEN技术可用于创建遗传突变体。使用RNAseq或基因表达的定量PCR也可以用于对某些宿主基因的表达表征响应于感染。

链球菌iniae是一个主要的水生病原体，其能够引起全身性疾病在两个鱼和人1。而S. iniae负责的水产养殖业巨大损失，这也是一个潜在的人畜共患的病原体，能够与类似的其他引起链球菌人类病原体的临床病症引起的疾病免疫功能低下的人类宿主。鉴于其相似性与人类的病原体，它来研究S.是非常重要的在天然宿主的上下文iniae疾病的发病机制。 S的 ​​成年斑马鱼模型iniae感染揭示宿主白细胞向感染的局部部位，以及一个快速时间来承载死亡健壮浸润，时间太短，涉及适应性免疫系统7。为了获得一个深入的研究，以S.先天性免疫应答iniae感染在体内 ，有必要使用一个模型，该模型是更适合为n对创实时成像。 幼虫斑马鱼具有许多优点，使得它越来越具有吸引力脊椎动物模型用于研究宿主 - 病原体相互作用。斑马鱼是相对便宜且易于使用和维护相比哺乳动物模型。适应性免疫并不功能成熟之前4-6周受精后，但幼虫具有高度保守的脊椎动物先天免疫系统与补体，Toll样受体，细胞因子，和嗜中性粒细胞和巨噬细胞具有抗微生物功能，包括吞噬作用和呼吸爆发2-6 8-11。此外，遗传易处理性和发展的胚胎和幼虫阶段的光学透明性允许对稳定的转基因品系的产生与荧光标记的免疫细胞使得能够检查实时体内宿主-病原体相互作用。使用photoconvertible蛋白质，这些转基因品系，如书房的产生dra2允许单个宿主细胞的起源和命运的超过12感染的过程中的跟踪。 当开发一个斑马鱼幼体感染模型，显微注射的选址将确定是否感染最初是局部或全身。全身血液感染入尾静脉或居维叶的导管是最常用的研究微生物病原体在斑马鱼和可用于研究在病原体菌株之间的毒力的主机和微生物细胞，细胞因子应答，和差之间的相互作用是有用的。对于生长较慢的微生物，早期注入的胚胎在16-1,000细胞阶段的卵黄囊可用于产生全身感染13,14，具有最佳的发育阶段对发现是间生长缓慢的微生物的显微注射在16到128细胞阶段15。但是，蛋黄许多微生物囊注射，以后主机的发展阶段往往是致命的吨他主持由于对白细胞浸润16-18的微生物和缺乏营养丰富的环境。 本地化感染通常会导致对感染的部位，可以很容易量化以非侵入性成像的白细胞的定向迁移。这种类型的感染可以允许介导白细胞迁移以及各种白细胞群体的不同迁移和吞噬能力调查机制的解剖。局部感染也是有用的研究中的细菌菌株的毒力之间的差异时，以及研究微生物入侵机制，因为物理主机的障碍必须越过为一个局部感染，成为全身。斑马鱼通常升高的温度下25-31℃的19，但它们也可以被保持在温度高达34-35℃的某些人类病原体侵袭的严格的温度要求的研究毒力20，21。 许多不同的位点已被用来产生一个初始局部细菌感染包括后脑心室22，背侧尾肌18，心包腔23和耳泡（耳）5，16，24。然而，已经发现，注射细菌进入尾肌可造成组织损伤的细菌中，当调查白细胞响应13可能扭曲的结果的和炎症独立。虽然少损伤与注入到相关的后脑，虽然它最初是在缺乏幼胚白细胞，后脑脑室稳步随着时间的推移获得更多的免疫细胞为小胶质细胞定居。后脑心室也是比较困难的位置到图像。耳囊是一个封闭的空腔，没有直接进入脉管25,26。它通常是缺乏列伊kocytes，但白细胞可以响应于炎症刺激如感染被招募到耳泡。它也是细菌微量注射在斑马鱼老化2-3天受精后，因为成像的易用性和喷射的可视化（dpf）的一个优选的位点。因此，我们选择了耳泡作为我们本地化细菌感染的部位。

<table><tbody><tr><td>1.7 ml Eppendorfs</td><td>MidSci</td><td>AVSS1700</td><td></td></tr><tr><td>14 ml Falcon tube</td><td>BD Falcon</td><td>352059</td><td></td></tr><tr><td>27 G x &amp;frac12; in. needle</td><td>BD Biosciences</td><td>305109</td><td></td></tr><tr><td>96-well Plate</td><td>Corning Incorporated</td><td>3596</td><td></td></tr><tr><td>Agar</td><td>BD Biosciences</td><td>214030</td><td></td></tr><tr><td>CellTracker Red</td><td>Molecular Probes, Invitrogen</td><td>C34552</td><td></td></tr><tr><td>CNA agar</td><td>Dot Scientific, Inc</td><td>7126A</td><td></td></tr><tr><td>Disposable transfer pipets</td><td>Fisher Scientific</td><td>13-711-7m</td><td></td></tr><tr><td>Dissecting scope</td><td>Nikon</td><td>SMZ745</td><td></td></tr><tr><td>DMSO</td><td>Sigma Aldrich</td><td>D2650</td><td></td></tr><tr><td>Ethanol 200 proof</td><td>MDS</td><td>2292</td><td></td></tr><tr><td>Fine tweezers</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>Gel comb</td><td>VWR</td><td>27372-482</td><td>4.2 mm width, 1.5 mm thick</td></tr><tr><td>Glass bottom dishes</td><td></td><td></td><td>Custom made by drilling a 16 - 18 mm hole in the center of a 35 mm tissue culture dish bottom and placing a 22 mm round #1 coverslip in the hole and sealing with a thin layer of Norland Optical Adhesive 68 cured by UV light.</td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>G33-4</td><td></td></tr><tr><td>High melt agarose</td><td>Denville Scientific, Inc.</td><td>CA3510-6</td><td></td></tr><tr><td>Hydrogen peroxide</td><td>Fisher Scientific</td><td>H325</td><td></td></tr><tr><td>Laser scanning confocal microscope</td><td>Olympus</td><td>with FV-1000 system</td><td></td></tr><tr><td>Low melt agarose</td><td>Fisher</td><td>BP165-25</td><td></td></tr><tr><td>Magnetic stand</td><td>Tritech (Narishige)</td><td>GJ-1</td><td></td></tr><tr><td>Microinjection system</td><td>Parker</td><td>Picospritzer III</td><td></td></tr><tr><td>Microloader pipet tips</td><td>Eppendorf</td><td>930001007</td><td></td></tr><tr><td>Micromanipulator</td><td>Tritech (Narishige)</td><td>M-152</td><td></td></tr><tr><td>Micropipette puller</td><td>Sutter Instrument Company</td><td>Flaming/Brown P-97</td><td></td></tr><tr><td>Nanodrop spectrophotmeter</td><td>Thermo Scientific</td><td>ND-1000</td><td></td></tr><tr><td>N-Phenylthiourea (PTU)</td><td>Sigma Aldrich</td><td>P7629</td><td></td></tr><tr><td>Paraformaldheyde</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>Petri dishes</td><td>Fisher Scientific</td><td>FB0875712</td><td>100 x 15 mm</td></tr><tr><td>Phenol</td><td>Sigma Aldrich</td><td>P-4557</td><td></td></tr><tr><td>Phenol Red</td><td>Ricca Chemoical Company</td><td>572516</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Fisher Scientific</td><td>BP665-1</td><td></td></tr><tr><td>Potassium hydroxide</td><td>Sigma Aldrich</td><td>P-6310</td><td></td></tr><tr><td>Pronase</td><td>Roche</td><td>165921</td><td></td></tr><tr><td>Protease peptone</td><td>Fluka Biochemika</td><td>29185</td><td></td></tr><tr><td>Small cell culture dish</td><td>Corning Incorporated</td><td>430165</td><td>35 x 10 mm</td></tr><tr><td>Sudan Black</td><td>Sigma Aldrich</td><td>S2380</td><td></td></tr><tr><td>Thin wall glass capillary injection needles</td><td>World Precision Instruments, Inc.</td><td>TW100-3</td><td></td></tr><tr><td>Todd Hewitt</td><td>Sigma Aldrich/Fluka Analytical</td><td>T1438</td><td></td></tr><tr><td>Tricaine (ethyl 3-aminobenzoate)</td><td>Argent Chemical Laboratory/Finquel</td><td>C-FINQ-UE-100G</td><td></td></tr><tr><td>Triton X-100</td><td>Fisher Scientific</td><td>BP151-500</td><td></td></tr><tr><td>Tween 20</td><td>Fisher Scientific</td><td>BP337-500</td><td></td></tr><tr><td>Yeast extract</td><td>Fluka Biochemika</td><td>92144</td><td></td></tr></tbody></table>

non-invasive imaging of the innate immune response in a zebrafish larval model of streptococcus iniae infection

水生病原体， 链球菌iniae，负责超过100亿美元的损失每年用于水产养殖业和能够引起全身性疾病在鱼类和人类。更好地理解S. iniae疾病的发病机制，需要一个合适的模型系统。遗传易处理性和斑马鱼的早期发育阶段的光学透明度允许对转基因品系的产生和非侵入性的成像用荧光标记的免疫细胞。适应性免疫系统不完全功能，直到数周后受精，但斑马鱼幼体有一个保守的脊椎动物先天性免疫系统与两个中性粒细胞和巨噬细胞。因此，幼虫感染模型的生成允许的先天免疫控制S.的具体贡献的研究iniae感染。 注射的部位将决定感染是否全身或局部开始。在这里，我们提出我们的协议耳泡注射的斑马鱼时效2-3天受精后，以及我们的技术感染荧光共焦成像。本地化感染部位可观察初始微生物入侵，招募宿主细胞和传播感染。使用S的斑马鱼幼体模型我们的研究结果iniae感染表明斑马鱼可用于检查主机嗜中性粒细胞和巨噬细胞在局部细菌感染的不同的贡献。此外，我们将描述如何免疫细胞photolabeling可用于感染过程期间跟踪单个宿主细胞的命运。

S的 ​​显微注射iniae进入耳泡（ 图1和图2）的结果的初始局部宿主反应。当正确地注入，细菌只应被看作在耳泡而不是在周围组织或血液。这可显微注射过程中使用的酚红染料（ 图1A）被可视化。另外，如果标记的细菌注射，感染幼虫的快速扫描立即注射后可以确认菌仅在耳泡而不是周围的组织（ 图1B）。虽然低至10 CFU野生型S.剂量iniae能够在24-48小时后的感染（HPI），注射1000 CFU的无毒菌株，CPSA的建立一个致命的感染，不会导致致死性感染和菌株似乎无法在主机24增殖。因此，这一局部感染模型能够分异改变致病的细菌菌株之间即最初局部感染的注射部位是研究白细胞趋化非常有用。白细胞募集可以通过苏丹黑嗜中性粒细胞颗粒染色（ 图1A）或荧光的转基因系（ 图1B）甲醛固定由定量。以可视化的招聘和免疫细胞的吞噬能力，我们使用表达绿色荧光蛋白转基因Dendra2线特别是在巨噬细胞中的Tg（MPEG1:dendra2）24 或中性粒细胞的Tg（MPX:dendra2）12。当S. iniae注入到3旦幼虫的耳泡，既嗜中性粒细胞和巨噬细胞的前2 HPI（ 图1）之内迅速招募。然而，当正确地进行穿孔，显微注射的PBS放入耳泡不会导致同一健壮招募宿主白细胞（ <strong&gt;的图1）。除了招聘，活的注射荧光标记的细菌都揭示了嗜中性粒细胞和巨噬细胞的吞噬能力，​​荧光的转基因系共焦时间推移成像。红色染料标记S. iniae既能嗜中性粒细胞和巨噬细胞（ 图2）内找到。使用photoconvertible蛋白Dendra2标记的中性粒细胞或巨噬细胞可用于非侵入性photolabeling对于跟踪单个细胞的命运在感染过程。被招募到耳泡在5 HPI的巨噬细胞，光转换，然后跟踪在接下来的24小时。虽然一些光转换单元保持在耳泡或头部区域，有些也可以发现在整个幼虫体内传播（ 图3）。 <img alt="图1" src="/files/ftp_upload/52788/52788fig1highres.jpg" width="550" /> <strong&gt;图1:白细胞招募到耳泡感染S. iniae。（A）中性粒细胞招聘S. iniae感染。 （ⅰ）成功注射酚红标记的接种物放入耳泡。 （ 二 - 四 ）苏丹黑幼虫染色中性粒细胞招聘2 HPI调查。 PBS模拟感染的幼虫秀有点中性粒细胞招募到耳泡（II），而感染野生型S. iniae或CPSA突变导致强大的中性粒细胞招聘（ 三 ， 四 ）。比例尺，300微米。 （B）巨噬细胞招聘S. iniae感染。 （I）的红色标记S.成功的显微注射iniae（品红色表示）到耳泡。 （ 二 - 四 ）显微注射转基因MP的荧光共焦图像EG1:dendra2幼虫固定在2 HPI。 PBS模拟感染的幼虫显示小巨噬细胞浸润（ii）中，但幼虫感染CellTracker红色标记（在品红描绘）野生型S. iniae或CPSA突变秀健壮的巨噬细胞招募到耳泡在2 HPI（三，四）。比例尺，30微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52788/52788fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52788/52788fig2highres.jpg" width="600" /> 图2:S.吞噬iniae通过在耳泡吞噬细胞的转基因MPX:dendra2（A）或MPEG1:dendra2（B）的幼虫感染红色标记S. iniae（在品红描绘）和使用激光scann成像，在60分钟后的感染荷兰国际集团共聚焦显微镜。比例尺，30微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52788/52788fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52788/52788fig3highres.jpg" width="700" /> 图3:巨噬细胞在耳泡5 HPI与S.光转化iniae。巨噬细胞（绿色描绘）在耳泡，由圆（A） 的指定，使用上的共焦显微镜405nm激光被光转换（B）和随时间跟踪。 24 HPI，光转换的巨噬细胞（以红色表示）迁移尽可能的主干/骶管造血组织（C）;比例尺，50微米。在C中的盒装区域的更高放大倍数示于（i）和（ii）中，比例尺条为30μm;箭头指向photocon巨噬细胞后来转换。因为合并后的543纳米的红色荧光，剩余的488 nm的绿色荧光的光转换细胞呈现白色。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52788/52788fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection at JoVE.com

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

先天免疫反应中的斑马鱼幼体型号无创成像<em&gt;链球菌iniae</em&gt;感染

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

non-invasive-imaging-innate-immune-response-zebrafish-larval-model

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

10.8K 次观看.  University of Wisconsin-Madison. Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle. 

视频: Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS expression system1 in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells. The KalTA4-ERT2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis2,3. Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.

Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae.

先天免疫和癌前细胞间的相互作用使用可诱导的Gal4 / UAS表达系统在斑马鱼幼体皮肤实时成像

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS ...

科研

发育生物学

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

斑马鱼的胚胎细胞内细菌病原体感染

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

免疫与感染

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

播散性念珠菌在斑马鱼幼虫的非侵入性成像

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique&#39;s relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

In this protocol, fluorescently labeled T. cruzi&#160;were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy.

斑马鱼幼虫动物模型的建立研究锥虫运动性体内

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

呼吸爆发的响应为先天免疫健康的斑马鱼中的指标量化

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions.&#160;The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model.&#160;In this respect, the present manuscript describes the use of zebrafish as an&#160;in vivo&#160;model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

破译和成像发病机制与盘带<em&gt;脓肿分枝杆菌</em&gt;斑马鱼胚胎

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United States. The innate immune response against C. difficile has been described, but the exact roles of neutrophils and macrophages in CDI are less understood. In the current study, Danio rerio (zebrafish) larvae are used to establish a C. difficile infection model for imaging the behavior and cooperation of these innate immune cells in vivo. To monitor C. difficile, a labeling protocol using a fluorescent dye has been established. A localized infection is achieved by microinjecting labeled C. difficile, which actively grows in the zebrafish intestinal tract and mimics the intestinal epithelial damage in CDI. However, this direct infection protocol is invasive and causes microscopic wounds, which can affect experimental results. Hence, a more noninvasive microgavage protocol is described here. The method involves delivery of C. difficile cells directly into the intestine of zebrafish larvae by intubation through the open mouth. This infection method closely mimics the natural infection route of C. difficile.

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Presented here is a safe and effective method to infect zebrafish larvae with fluorescently labeled anaerobic C. difficile by microinjection and noninvasive microgavage.

为梭状体开发拉瓦尔斑马鱼感染模型

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.

使用人类流感病毒感染的斑马鱼模型到屏幕抗病毒药物和表征宿主免疫细胞反应

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major pathogens in BAI. Current experimental BAI mammalian animal models such as mouse models are costly and time-consuming, and therefore not suitable for high throughput analysis. Thus, novel animal models as complementary systems for investigating BAI in vivo are desired. In the present study, we aimed to develop a zebrafish embryo model for in vivo visualization and intravital analysis of bacterial infection in the presence of biomaterials based on fluorescence microscopy. In addition, the provoked macrophage response was studied. To this end, we used fluorescent protein-expressing S. aureus and transgenic zebrafish embryos expressing fluorescent proteins in their macrophages and developed a procedure to inject bacteria alone or together with microspheres into the muscle tissue of embryos. To monitor bacterial infection progression in live embryos over time, we devised a simple but reliable method of microscopic scoring of fluorescent bacteria. The results from microscopic scoring showed that all embryos with more than 20 colony-forming units (CFU) of bacteria yielded a positive fluorescent signal of bacteria. To study the potential effects of biomaterials on infection, we determined the CFU numbers of S. aureus with and without 10 &#181;m polystyrene microspheres (PS10) as model biomaterials in the embryos. Moreover, we used the ObjectJ project file &#34;Zebrafish-Immunotest&#34; operating in ImageJ to quantify the fluorescence intensity of S. aureus infection with and without PS10 over time. Results from both methods showed higher numbers of S. aureus in infected embryos with microspheres than in embryos without microspheres, indicating an increased infection susceptibility in the presence of the biomaterial. Thus, the present study shows the potential of the zebrafish embryo model to study BAI with the methods developed here.

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

The present study describes a zebrafish embryo model for in vivo visualization and intravital analysis of biomaterial-associated infection over time based on fluorescence microscopy. This model is a promising system complementing mammalian animal models such as mouse models for studying biomaterial-associated infections in vivo.

一种用于生物技术相关金黄色葡萄球菌感染活体可视化和内膜分析的斑马鱼胚胎模型

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major ...

生物工程

Photoconversion-Based Leukocyte Tracking in Bacterial-Infected Transgenic Zebrafish

Source: Harvie, E. A. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52788">Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection.</a>&#160;J. Vis. Exp. (2015)
This video demonstrates the tracking of leukocyte recruitment using transgenic zebrafish larvae expressing photoconvertible green fluorescent protein. Pre-injecting red fluorescent dye-labeled bacteria into the otic vesicle and inducing photoconversion enables observation of leukocytes&#39; migration, phagocytosis, and dispersal throughout the body, providing valuable insights into immune response dynamics.

细菌感染的转基因斑马鱼中基于光转化的白细胞追踪

1. Preparing Microinjection Needles

	Prepare thin wall glass capillary injection needles (1.0 OD/0.75 ID) using a micropipette puller device with the ...

先天免疫反应中的斑马鱼幼体型号无创成像

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