作者感谢K. Kissa有益的讨论，并提供脂，氯膦酸二钠和L.莱玛克里斯南的pTEC27和pTEC15，让tdTomato和芥末的表现，分别的一份厚礼。这项工作形成了法国国家研究署的项目（ZebraFlam ANR-10-MIDI-009和DIMYVIR ANR-13-BSV3-007-01）和欧盟第七框架计划的一部分（FP7-人2011年ITN）根据赠款协议没有。 PITN-GA-2011-289209的玛丽 - 居里的初始培训网络FishForPharma。我们还要感谢该协会格雷戈里Lemarchal和Vaincre拉Mucoviscidose（RF20130500835），用于资助CM杜邦。

斑马鱼实验步骤必须符合相关的制度和政府规章。在本研究中，斑马鱼实验在大学蒙彼利埃，根据用于处理实验动物的欧盟指导方针（http://ec.europa.eu/environment/chemicals/lab_animals/home_en.htm），并在参考批准CEEA-LR-13007。 1.制备试剂和显微注射设备<ol><li>制备鱼水由在1升蒸馏水中溶解31 0.06克即时海洋海盐，然后高压灭菌消毒（120℃，20分钟），并存储在28.5℃下长达1个月。 </li><li>通过将1g亚甲蓝于1L蒸馏水制备亚甲蓝溶液。加入300微升亚甲蓝溶液在1L水中的鱼，获得蓝鱼水，然后高压灭菌消毒，并储存在28.5℃下长达1个月。 注:additi对亚甲基蓝鱼水可以防止霉菌生长。 </li><li>磷酸盐缓冲盐水的制备（PBS）中<ol><li>通过将5.696克的 Na 2 HPO 4，680毫克KH 2 PO 4，969毫克KCl和七十八点八九四克NaCl在1L蒸馏水制备一个10×PBS原液和将pH调节至7.0，用HCl。以获得1×PBS中，在20分钟内稀释百毫升在900ml蒸馏水和高压釜中的10×PBS溶液在120℃灭菌。 </li><li>添加0.05％吐温80​​，以获得1×PBST和存储在RT长达1年。 </li></ol></li><li>通过溶解8.5克氯化钠，加入50克牛血清白蛋白，将20g D-葡萄糖，40毫克过氧化氢酶从牛肝脏和0.5克油酸的在1升蒸馏水中，然后过滤灭菌溶液制备油酸葡萄糖过氧化氢酶（OADC）富集和它在4℃下储存长达6个月。 </li><li>制备100ml的3-氨基苯甲酸乙酯甲磺酸（三卡因）原液，通过将400毫克tricaiね粉末在97.9 ml的蒸馏水。加2.1毫升的1M Tris（pH为9）以调节pH在7.0和存储所述溶液在-20℃。 </li><li>通过拉动使用微量牵引装置具有以下设置硼硅玻璃毛细管（1毫米外径×0.78毫米的ID）制备微细管注射针:空气压力650;热火999;拉50;速度80;时间200。 </li><li>准备在蓝色的鱼水1.5％琼脂溶液，倒入100毫米培养皿25毫升融化的琼脂。在融化的琼脂的顶部，放置自制模具以压印特定频道。 "V"形通道用于位置胚胎而形用于蛋（图1）31的"U"形。一旦凝固，小心地取出印记。 注:盖上蓝色的鱼水琼脂，以避免脱水，并储存在4℃下长达2个月。 </li></ol><img alt="图1" src="/files/ftp_upload/53130/53130fig1.jpg"/&gt; 图1.定位室斑马鱼注射。 （一）鸡蛋（左图）和V形通道的胚胎（右图）的U形通道。斑马鱼卵子/胚胎奠定了在通道和沿同一轴线对齐。（B）可视腔的显微观察。 <a href="https://www.jove.com/files/ftp_upload/53130/53130fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 2.准备和M的存储脓肿接种<ol><li> 脓肿分支杆菌的生长条件<ol><li>盘开出M.脓肿从-80℃甘油到含有OADC的10％和0.5％的甘油（7H10 OADC）和补充有合适的抗生素取决于通过编码荧光蛋白的载体携带的抗性标记的米德尔7H10琼脂上。孵育板在30℃下4-5天。 </li><li>拿起荧光阳性分枝杆菌菌落，接种1 ml的7H9的Middlebrook肉汤补充有OADC，0.2％甘油，0.05％吐温80（7H9 OADC / T）用在15ml的无菌塑料管所需的抗生素。孵育在30℃下1周无晃动。 注:吐温80制约聚集和细菌聚集。 </li><li>重悬在2.1.2中获得的预培养在50ml的Middlebrook 7H9 OADC / T 150厘米2组织培养瓶中至最终0.1的OD 600（对应于围绕5.10 7细菌/ ml），并进一步孵育在30℃下进行4天，得到的指数增长的培养物（OD 600 = 0.6至0.8）。 注:为了尽量减少聚集，尤其是粗糙的变种，孵化不应超过5天。光滑和粗糙的变体显示了类似的增长速度。 </li></ol></li><li> 分枝的制备脓肿接种60; 注:由于毛坯M的高倾向脓肿形成大的聚集体，并产生线，特定的治疗是必要的，以之前在胚胎显微注射产生均匀且定量地控制接种物。该处理也适用于平滑产生聚集体，尽管以比粗应变程度较轻的细菌。 <ol><li>收获自150厘米2组织培养瓶中通过离心指数生长培养物在4，000×g离心在RT 15分钟在50ml无菌塑料管和悬浮细菌沉淀在1ml 7H9 OADC / T。分装200微升菌悬液到1.5ml微量管。 注意:小体积的工作在1ml注射器必须获得高度均匀的悬浮液。 </li><li>均化细菌悬浮液用26-G针（15向上和向下序列），超声处理三次，持续10秒（每个超声处理本身之间10秒休息组成的序列）使用超声波仪水浴。加入1毫升7H9 OADC / T和涡简要介绍。离心3分钟，在100×g下。 </li><li>小心收集含分枝杆菌的上清液，以避免团块和凝聚均匀悬浮在50ml的无菌塑料管。 </li><li>目视检查，以便最终剩余细菌聚集体的存在。 注:如果聚集体仍然存在，则继续执行一个附加的离心步骤，在100×g离心2分钟，回收上清。 </li><li>离心悬浮液在4000 XG 5分钟，悬浮颗粒在200微升7H9 OADC / T，然后继续注射。 </li><li>通过电镀系列稀释（在1×PBST中）上7H10琼脂OADC和后4天孵化计数菌落形成单位（CFU）在30℃下评估的最终细菌浓度。的CFUs应为每个新批次来确定。 </li><li>通过存储5微升等份在-80＆＃制备冷冻菌剂股176℃。 注:在-80℃冷冻等分试样的CFU评估是一个先决条件，以确定活菌的确切数目作为冷冻/解冻可能影响接种细菌存活。这些冷冻接种准备用于随后的注射。由于所有等份包含CFU的数相同，它们允许从一个实验到另一个注入细菌中可重复的方式。 </li></ol></li><li>接种质量控制 注:抗酸染色（特定于分枝杆菌），可以用于将细菌悬浮液的质量的前向和后在2.2中描述的均化步骤进行比较。 <ol><li>现货和摊开均质菌悬液在载玻片上的一滴并使其完全干燥，解决了拖影为至少30分钟的热板设定为65-70℃，染色用抗酸染色方案。 </li><li>观察细菌涂片用显微镜用100X目标和与非处理的细菌悬浮液的涂抹比较（ 图2）。 </li></ol></li></ol><img alt="图2" src="/files/ftp_upload/53130/53130fig2.jpg" /> 图2.制备分散M的脓肿菌剂。R或S的抗酸染色变体之前的任何处理（上图）上生长肉汤培养基或处理（下图）后，以减少分支杆菌聚集体的大小和数量（连续步骤syringing，超声处理和倾析） 。使用具有100×APO油1.4 NA物镜）显微镜细菌进行观察。比例尺:20微米<a href="https://www.jove.com/files/ftp_upload/53130/53130fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 3.准备斑马鱼胚胎<ol><li>产卵和收集斑马鱼的卵<ol><li>前S中的一天典当，通过将2男1女品种成年斑马鱼（通常是2:1的比例提供了最佳的受精率）进入养殖室，包括一个独立的鱼缸有一个鸡蛋收集篮31。 注:鸡蛋收集筐被用来保护鸡蛋被吃掉的父母和促进卵子收获。 </li><li>在1 HPF，丰年虫卵，只有正确地开发的胚胎在100毫米的培养皿在28.5℃，填充25毫升蓝色的鱼水（每盘最多100个胚胎），并留住他们。非受精卵被丢弃。 </li></ol></li><li>斑马鱼胚胎注射的制备<ol><li>在约24小时受精后（HPF），dechorionate用细镊子胚胎在100毫米的培养皿并保持在28.5℃。 </li><li>收集用吸管的30-48 HPF胚胎，并将它们传送到一个​​"V"形的定位腔室填充有含有25 ml的鱼水的270毫克/升三卡因。使用自制的微加载提示工具（自制修剪微加载提示）一个更简单的微操作优化的渠道妥善奠定胚胎。 注:向下定向背侧在尾静脉或背侧向上的尾肌注射静脉注射。 </li></ol></li></ol> 4.微注射程序注意:微注射程序分枝脓肿类似于前面分枝描述的海鱼打针32。为了评估M的年表脓肿感染（生存，细菌负载，动力及感染的特征），在30 HPF胚胎尾静脉注射是首选。为了显现免疫细胞的招聘，注射完成本地化的网站，如在尾部的肌肉在48 HPF胚胎。 <ol><li>稀释在1×PBST（分枝杆菌接种物取决于CFU与数给予如步骤2.2.6）确定，含10％酚红来检查适当的注入。 </li><li>加载微毛细管注射针用5-10微升细菌接种物使用微加载尖端，连接显微注射针进入三维显微连接到所述喷射器的夹持器和断绝与细镊子针的顶部，以获得5-10微米开口直径。 </li><li>通过调整显微注射压力（20-50帕斯卡）和时间（0.2秒）校准注射体积。 注意:需要注射体积由视觉确定排出成胚胎的蛋黄的液滴的直径的校准。 </li><li>对于尾静脉注射，定位与腹侧朝向所述针（如在3.2.2节中描述）的胚胎，然后将针尖接近泌尿开口，靶向尾静脉，并轻轻针尖推入胚胎直到它刚好刺穿caud人脉地区。递送细菌制剂的所需体积，一般1-3 NL含有约100单位/ NL。 </li><li>对于肌内注射，定位与背侧朝向所述针（如在部分3.2.2）的胚胎，将针通过一个体节和注入接种小体积（1 NL）。 注:对于尾静脉注射，肌肉注射感染也很具有挑战性的执行。必须小心，以避免可能伤害周围组织过载。 </li><li>在注射过程结束时，通过注射无菌的PBST下降，随后通过电镀在7H10 OADC和计数的CFU的细菌悬浮液的相同体积控制接种物的大小。大约100胚胎可以用针注射。 注:由于毛坯M.脓肿可继续以形成聚集体中的针，注射液需要被制备接种物后立即进行。 </li><li>转移感染FISħ单独在24孔板含2ml /孔鱼水和孵化在28.5℃。胚胎注射1-3 NL 1×PBST中可以用作对照。 </li><li>适当的感染荧光细菌的胚胎是使用荧光显微镜监测。 </li></ol> 5.生成巨噬细胞耗尽的胚胎注:从组织的巨噬细胞的选择性消耗来调查他们的感染过程中的贡献和作用。为了显现巨噬细胞的适当的耗尽时，建议使用具有荧光的巨噬细胞，其中mCherry是专门的巨噬细胞特异性启动子的mpeg1 19的控制下表达的转基因系。 <ol><li>前列地氯膦酸盐为基础的程序 注:脂，氯膦酸二钠程序允许巨噬细胞从组织选择性耗尽（但无中性粒细胞）19。这种化合物既不能改变的胚胎，也没有自动对焦的生存FECT细菌的完整性。一份详细的协议，准备脂质体包裹氯膦酸二钠先前已33报道。 <ol><li>在24 HPF，dechorionate胚胎，并将它们传送到一个​​"V"形注射菜填充有补充有三卡因如3.2.2所述，保持在麻醉状态下的胚胎直到注射过程结束时，鱼水。向下定向背侧。 </li><li>加载微细管的注射针，用脂质体包封的氯膦酸盐（脂-氯膦酸盐），连接显微注射针进入三维显微的保持器和断绝与细镊子针的顶部，以获得10μm的前端开口的直径。 </li><li>要校准的注射量，调整显微注射压力到20百帕和喷射时间，以0.2秒。将针尖接近泌尿开口，靶向尾静脉，并轻轻针尖推入胚胎直到它只是刺穿尾静脉区和提供的溶液的所需体积（2-3 NL，3次）。 注:脂，氯膦酸二钠悬挂很粘，并在注射前应涡旋以避免阻塞胚胎的血管系统和杀戮。关键是要着手小体积几个连续注射。 </li><li>胚胎转移中含有的鱼水100 25mm培养皿让他们在28.5°C，直到感染。 </li><li>控制巨噬细胞荧光显微镜适当的枯竭。 注:完整的巨噬细胞耗竭是有效的脂-clodoronate给药后4天。 </li></ol></li><li>吗啉基程序<ol><li>产卵，繁殖成年斑马鱼的前一天，通过将2男1女在育种室的塑料屏障隔开。 </li><li>第二天，≈30分钟后，在斑马鱼设施指示灯亮起，拆除障碍分离男性和女性。等待产卵开始后约20分钟，以优化的受精率。收获的鸡蛋，通过转移他们在100毫米培养皿中充满了寒潮蓝色的鱼水（4℃）减缓其发展。 注:产卵的控制是为吗啉基实验的关键。吗啉代只能被注入到蛋多达四个单元的阶段。 </li><li>收集用吸管将鸡蛋和沉积卵倒在"U"形的注射室填充有冷蓝色鱼水，并用自制的微加载前端工具方框在通道中的卵。 </li><li>制备含有赤苯酚10％的PU.1吗啉溶液，如前所述19。加载微毛细管注射针与吗啉制备，并连接显微注射针进入三维显微的保持器，然后断绝与细镊子针的尖端obtai5-10微米呐顶部开口的直径。 </li><li>要校准的注射量，调整显微注射压力到20-50百帕和喷射时间，以0.2秒。将针尖接近蛋并轻轻通过绒毛膜推针的前端到蛋和提供所需的吗啉溶液体积，通常为3-5 NL。 </li><li>显微注射后，在鱼水转移鸡蛋100毫米培养皿和孵化在28.5℃，直到感染的程序开始。 </li><li>在30〜48高倍视野分析正确的（完全）的PU.1 morphants巨噬细胞的荧光显微镜枯竭。 注意:根据注入的浓度，对U.1吗啉也可以影响早期嗜中性粒细胞的数量。以确认巨噬细胞的正常消耗而不改变嗜中性粒细胞的数量时，建议使用具有荧光的巨噬细胞和嗜中性粒细胞的双重转基因斑马鱼线（用GFP由MPX启动专门驱动的表达）19。 </li></ol></li></ol> 6.细菌负荷定量<ol><li>测定CFU枚举<ol><li>在期望的时间点，收集感染的胚胎在1.5ml微离心管（每条件5）基团（1胚胎/管），低温麻醉胚胎通过在冰上温育10分钟，用安乐死三卡因的过量（300- 500毫克/升） </li><li>洗用无菌水将胚两次和分配它们在一个新的管中，裂解每个胚，用2％的Triton X-100使用26-G针和均化的悬浮液直到完全裂解稀释在1×PBST。离心悬浮液和重悬沉淀在1×PBST含0.05％Tween的匀浆80.系列稀释液铺于7H10米德尔OADC和补充有BBL MGIT PANTA，所推荐的供应商。 注:M。脓肿是更容易的NaOH treatmen牛逼不是M.鱼裂解物，而不会影响海分枝杆菌，去污脓肿生长可使用BBL MGIT PANTA抗生素鸡尾酒成功地实现。 </li><li>孵育所述板4天，在30℃和计数菌落。 </li></ol></li><li>测定荧光像素计数活胚胎（FPC）的测量 注:确定通过荧光细菌负荷，受感染的胚胎序列图像采集，并通过FPC（细菌鉴定通过颗粒分析）测量荧光强度使用的ImageJ的免费软件开发的国产宏。一种粒子分析需要的二进制黑白图像，依赖于一个阈值范围，允许鉴别的从背景感兴趣的荧光信号。 <ol><li>在期望的时间点，如在3.2.2中描述tricain-麻醉感染胚胎（每种条件5-10）35毫米培养皿中。 </li><li>除去水和次亚RGE胚胎和整个盘表面与鱼水1％低熔点琼脂糖，然后对齐横向胚胎。覆盖凝固的琼脂糖含三卡因鱼水。 </li><li>获取使用落射荧光显微镜用10×的目标与整个胚胎的荧光图像。 注:荧光图像采集是在FPC测量过程的一个关键步骤。使用经过校准的显示器用彩色校准探针是很重要的。调节曝光的最佳时间，以获得最小的背景期间采集防止信号的饱和度。图像必须是一个8位的TIFF格式。相同的设置是在整个实验进行定量的目的过程中保持。 </li><li>从用自制的微加载尖端工具的胚胎小心地取出琼脂糖。这些胚胎可以用于随后的分析，如果必需的。在此之前的细菌负荷的量化，每个画面应质量控制。要在nalyze图像和转换荧光强度为每FPC鱼，打开ImageJ的免费。 注:FPC值反映了细菌的负担，因为以前使用M.所示海鱼34。 </li><li>确定用于图像分析所需的阈值使用未感染的胚胎的图像（数对照胚胎可以用来得到一个平均的阈值）。转到图像→调整→阈值，然后移动底部滑块的门槛右侧窗口，直到背景变成全黑（ 即获得背景应该是零）。记录对应的值。 </li><li>在的Image J，打开FPC宏（补充文件），并按照提示:定位要测量的图像的文件夹，然后进入门槛为先前确定的，并点击"确定"。 注:宏自动减去背景。阈值，然后应用。荧光信号鉴定田间以下的颗粒分析。 </li><li>复制并从摘要窗口中的数据粘贴到数据分析所需的软件。 注意:在此相反的CFU确定方法，将FPC方法是非侵入性的，并允许重复使用胚胎随后的分析，并且重要的是，监测动力学/的个别细菌负荷动力学。因此，它允许显著减少动物的数量，在与伦理准则协议。 </li></ol></li></ol> 7.成像M. abscessus-感染胚胎<ol><li> M的实时成像脓肿感染<ol><li>摩三卡因麻醉胚胎（见3.2.2）中之前，落射荧光显微镜观察在35毫米陪替氏培养皿1％低熔点琼脂糖。使用玻璃底培养皿倒置共聚焦显微镜或在一个单腔凹陷滑动直立共聚焦显微镜。定向胚胎到所需的位置和盖凝固的琼脂糖含三卡因鱼水。 </li><li>使用落射荧光显微镜用整个胚胎的连续荧光采集和传输图像的10×物镜。另外，使用共焦显微镜，40X或63X目标的感染后的可视化的免疫细胞的活性。 </li></ol></li><li>影像固定M.脓肿感染胚胎<ol><li>安乐死胚胎如6.1.1所述和他们在1×PBST进行2小时，在室温或O / N在4℃下转移到1.5 ml的微离心管中并固定在胚胎4％多聚甲醛。洗涤胚胎两次，用1×PBST 10分钟除去多聚甲醛。 注:为了保护荧光，固定的胚胎必须避光保存。 </li><li>为了保存组织和荧光的完整性，胚在增加甘油浓度的溶液（10，20，30，40和50％的稀释在1×PBST中）依次培养，用于每种条件10分钟。 注:固定的胚胎可存放数个月在50％甘油，4℃。 </li><li>躺在嵌入在50％甘油中观察室（图1B）31中的胚胎。 </li><li>图片使用40X或63X的目标上共聚焦显微镜。 </li></ol></li></ol>

The authors have nothing to disclose.

斑马鱼最近出现作为优良的脊椎动物模型系统用于使用实时36广阔的领域，共焦成像研究细菌感染的动态。分散分枝杆菌悬浮液（协议2.2）与显微注射方法（协议4）相结合，可重现的全身性感染，以及随后的监测和感染，特别注重与主机巨噬细胞细菌相互作用的进展可视化。 M的毒力脓肿体内可以调查由于使用的野生型金黄斑马鱼37。为了研究M之间的相?...

脓肿分枝杆菌是一种新兴的病原体，导致广泛的人类临床综合征谱。这些包括皮肤感染以及严重的慢性肺部感染，免疫功能低下和囊性纤维化患者1,2,3,4大多遇到。M.脓肿也算是一大快速增长的分枝杆菌负责人和医院和医源性感染的物种。此外，最近的几份报告强调了可能性，即M.脓肿可以穿过血-脑屏障并诱导在中枢神经系统（CNS）5,6-重要病变。尽管是一个快速的种植者，M。脓肿展品还与相关的那些结核分枝杆菌的致病性的几个功能，包括肉芽肿结构中保持沉默多年，产生干酪样病变的肺部7的能力。更令人震惊的是低仙M的敏度脓肿抗生素，使这些感染极难治疗导致显著治疗失败率8,9。这一物种的重要威胁，主要是其对抗生素的内在阻力，这是公共卫生机构10重大关切和禁忌肺移植11。 脓肿分支杆菌显示平滑（S）或粗（R）的菌落形态，导致不同临床结果。与此相反的S品系，R细菌有增加的趋势端到端，导致绳索或条索状结构12,13。基于两种细胞或动物模型有几个独立的研究揭示了R的超毒力型形态型14,15。从流行病学研究，M的最严重的情况下， 脓肿肺部感染似乎被R相关联的变体16，它是唯一的变体一直被视为持续数年被感染主机 3。该形态类型的差异依赖于表面相关glycopeptidolipids（GPL）12的存在（在S）或损失（在读）。然而，由于目前可用的蜂窝/动物模型的固有局限性用于研究M.脓肿感染，我们对R或S变体的病理生理事件的知识仍然是模糊的。 通过静脉注射或气溶胶途径免疫能力的小鼠感染导致短暂的殖民统治，阻碍利用老鼠来研究持续感染和体内药物敏感试验17。因此，开发的动物模型适合于宿主反应的操作是一项​​重大的挑战。在这种背景下，近来已经开发感染的非哺乳动物模型，包括果蝇 18，提供了几个优点，例如成本，速度和伦理可接受ö版本1.00的小鼠模型。斑马鱼（ 斑马鱼 ）感染模型也已经探索了可视化，通过无创成像，M的进展和年表脓肿感染的活的动物19。重要的是，概念证明也成立了证明其适合在体内对M.抗生素评估脓肿 17,20。 斑马鱼已经在过去二十年期间已广泛用于研究各种病原体和宿主的免疫系统 21之间的交互。这种替代脊椎动物模式的日益成功，依赖于激励和验证其用于更好地了解众多的病毒和细菌感染19,22,23,24,25,26,27,28,29重大而独特的机会。相对于大多数其他的动物模型，斑马鱼的胚胎是光学透明的，从而允许非侵入性荧光成像30。此HA为首的研究M.脓肿感染斑马鱼的胚胎以前所未有的细节，与细胞外录制的说明中，表示细菌形态可塑性的一例最终。盘带代表免疫系统颠覆的新机制和一个关键机制促进急性M的发病机制脓肿感染19。 这份报告描述了使用斑马鱼胚胎破译M的病理生理特点新的工具和方法脓肿感染并研究杆菌和先天免疫系统之间的亲密互动。首先，将详细显微注射协议，它包括的细菌接种物，胚胎制备，和本身感染的处理，提出。方法特别适用于评估M.脓肿毒力通过测量各种参数，如主机存活和细菌负荷，现介绍。特别重点就如何进行监控，在时空水平，感染的命运和进展以及为 M.宿主的免疫反应脓肿利用视频显微镜。此外，为了调查M.期间的贡献和巨噬细胞的作用脓肿感染，方法生成的巨噬细胞的贫胚胎（使用genetically-或化学为基础的方法）进行说明。最后，协议的可视化使用固定的或活的胚胎的巨噬细胞和中性粒细胞记录的特定交互。 本报告的目的是刺激进一步的研究阐明新的光转化为M.脓肿毒力机制和盘带在建立急性和不受控制的感染过程的特别的作用。

<table><tbody><tr><td>BBL MGIT PANTA</td><td>BD Biosciences</td><td>245114</td><td></td></tr><tr><td>Bovine Serum Albumin&amp;nbsp;</td><td>Euromedex</td><td>04-100-811-E</td><td></td></tr><tr><td>Catalase from Bovine Liver&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C40</td><td></td></tr><tr><td>Difco Middlebrook 7H10 Agar</td><td>BD Biosciences</td><td>262710</td><td></td></tr><tr><td>Difco Middlebrook 7H9 Broth</td><td>BD Biosciences</td><td>271310</td><td></td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate salt (Tricaine)</td><td>Sigma-Aldrich</td><td>A5040</td><td></td></tr><tr><td>Oleic Acid</td><td>Sigma-Aldrich</td><td>O1008</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Delta Microscopie</td><td>15710</td><td></td></tr><tr><td>Phenol Red</td><td>Sigma-Aldrich</td><td>319244</td><td></td></tr><tr><td>Tween 80</td><td>Sigma-Aldrich</td><td>P4780</td><td></td></tr><tr><td>Agar</td><td>Gibco Life Technologie</td><td>30391-023</td><td></td></tr><tr><td>Low melting agarose</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Instant Ocean Sea Salts&amp;nbsp;</td><td>Aquarium Systems Inc</td><td></td><td></td></tr><tr><td>Borosilicate glass capillaries&amp;nbsp;</td><td>Sutter instrument Inc</td><td>BF100-78-10</td><td>1 mm O.D. X 0.78 mm I.D.</td></tr><tr><td>Micropipette puller device&amp;nbsp;</td><td>Sutter Instrument Inc</td><td></td><td>Flamming/Brown Micropipette Puller p-87</td></tr><tr><td>Microinjector</td><td>Tritech Research&amp;nbsp;</td><td></td><td>Digital microINJECTOR, MINJ-D</td></tr><tr><td>Tweezers</td><td>Sciences Tools inc</td><td></td><td>Dumont # M5S&amp;nbsp;</td></tr><tr><td>Microloader Tips</td><td>Eppendorf</td><td></td><td></td></tr></tbody></table>

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.

虽然各种解剖部位可以注入32，尾静脉注射经常被用来产生全身感染的后续分析，包括存活实验中，细菌负荷判定，吞噬活性或帘线形成。在尾部肌肉注射用于评估的巨噬细胞的募集在注射（图3A）的部位。为了研究和比较M的 R和S变种的毒力脓肿 ，荧光细菌悬浮液注入在30 HPF胚胎（ 图3A）19 的尾静脉。与此相反的S变体时，R形态型诱?...

Watch this Scientific Journal Video about Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos at JoVE.com

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 ...

deciphering-imaging-pathogenesis-cording-mycobacterium-abscessus

Research

JoVE Journal

Immunology and Infection

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

10.1K 次观看.  Université Montpellier. 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. 

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

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

在活斑马鱼胚胎成像的亚细胞结构

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

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 and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

建立和高通量安装程序研究优化<em&gt;表皮葡萄球菌</em&gt;和<em&gt;分枝杆菌</em&gt;感染作为模型药物发现

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

Displacement Analysis of Myocardial Mechanical Deformation (DIAMOND) Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to reliably detect segmental mechanics and are not readily feasible in zebrafish. Here we present a semiautomated, open-source method for the quantitative assessment of four-dimensional (4D) segmental cardiac function: displacement analysis of myocardial mechanical deformation (DIAMOND). Transgenic embryonic zebrafish were imaged in vivo using a light-sheet fluorescence microscopy system with 4D cardiac motion synchronization. Acquired 3D digital hearts were reconstructed at end-systole and end-diastole, and the ventricle was manually segmented into binary datasets. Then, the heart was reoriented and isotropically resampled along the true short axis, and the ventricle was evenly divided into eight portions (I&#8211;VIII) along the short axis. Due to the different resampling planes and matrices at end-systole and end-diastole, a transformation matrix was applied for image registration to restore the original spatial relationship between the resampled systolic and diastolic image matrices. After image registration, the displacement vector of each segment from end-systole to end-diastole was calculated based on the displacement of mass centroids in three dimensions (3D). DIAMOND shows that basal myocardial segments adjacent to the atrioventricular canal undergo the highest mechanical deformation and are the most susceptible to doxorubicin-induced cardiac injury. Overall, DIAMOND provides novel insights into segmental cardiac mechanics in zebrafish embryos beyond traditional ejection fraction (EF) under both physiological and pathological conditions.

Displacement Analysis of Myocardial Mechanical Deformation DIAMOND Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

The goal of this protocol is to detail a novel method for the assessment of segmental cardiac function in embryonic zebrafish under both physiological and pathological conditions.

心肌机械变形（DIAMOND）的位移分析揭示了胚胎斑马鱼心脏功能的分段异质性

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to ...

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

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

marinum 分枝杆菌感染成人斑马鱼结核的建模

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune system during early development. The Zebrafish Mycobacterium marinum (M. marinum) infection model has been well-established in studying host immune response against mycobacterial infection. It has been suggested that different macrophage cell death types will lead to the diverse outcomes of mycobacterial infection. Here we describe a protocol using intravital microscopy to observe macrophage cell death in zebrafish embryos following M. marinum infection. Zebrafish transgenic lines that specifically label macrophages and neutrophils are infected via intramuscular microinjection of fluorescently labeled M. marinum in either the midbrain or the trunk. Infected zebrafish embryos are subsequently mounted on low melting agarose and observed by confocal microscopy in X-Y-Z-T dimensions. Because long-term live imaging requires using low laser power to avoid photobleaching and phototoxicity, a strongly expressing transgenic is highly recommended. This protocol facilitates the visualization of the dynamic processes in vivo, including immune cell migration, host pathogen interaction, and cell death.

This protocol describes a technique for visualizing macrophage behavior and death in embryonic zebrafish during Mycobacterium marinum infection. Steps for the preparation of bacteria, infection of the embryos, and intravital microscopy are included. This technique may be applied to the observation of cellular behavior and death in similar scenarios involving infection or sterile inflammation.

通过生命内显微镜在斑马鱼胚胎的分孔细菌感染过程中巨噬细胞细胞死亡的可视化

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune ...

<meta charset="utf8"></head><body>阐明斑马鱼视盖发育中的小胶质细胞行为

Live Imaging and Characterization of Microglia Dynamics in the Zebrafish Embryo

Microglia are highly dynamic cells and their migration and colonization of the brain parenchyma is a crucial step for proper brain development and function. Externally developing zebrafish embryos possess optical transparency, which along with well-characterized transgenic reporter lines that fluorescently label microglia, make zebrafish an ideal vertebrate model for such studies. In this paper, we take advantage of the unique features of the zebrafish model to visualize the dynamics of microglia cells in vivo and under physiological conditions. We use confocal microscopy to record a timelapse of microglia cells in the optic tectum of the zebrafish embryo and then, extract tracking data using the IMARIS 10.0 software to obtain the cells' migration path, mean speed, and distribution in the optic tectum at different developmental stages. This protocol can be a useful tool to elucidate the physiological significance of microglia behavior in various contexts, contributing to a deeper characterization of these highly motile cells.

<meta charset="utf8"></head><body>作者聚焦：使用 Zebrafish 探索大脑发育过程中的小胶质细胞迁移

We demonstrate a method that takes advantage of fast scanning confocal microscopy to perform live imaging of microglia cells in the developing zebrafish optic tectum, allowing for the analysis of the dynamics of these cells in vivo.

破译和成像发病机制与盘带

摘要

探索更多视频

此视频中的章节