笔者想感谢显微注射培训，克拉丽莎亨利的意见，对加快胚胎发育和设备的使用博士卡罗尔金的实验室，和，弥敦道劳森贡献FLI1:EGFP鱼。我们感谢手稿的批判性阅读的惠勒实验室和肖恩墙成员。我们也想感谢马克Nilan鱼的保健和咨询，瑞安Phennicie和克里斯汀的Gabor在这个项目上的技术咨询。资助这项工作是由MAFES哈奇授予1 MAFES E08913-08，和国立卫生研究院的NCRR奖P20RR016463河惠勒光兄弟，研究助理。

所有斑马鱼的护理协议和实验机构动物照顾及使用委员会（IACUC）协议A2009-11-01下进行。 1。吗啉和幼虫注射菜 实验时间:*（10-15分钟） 难度:* <ol><li>对于鸡蛋打针，准备在无菌水和微波的2％的琼脂糖溶液。当溶液冷却，倒入了一些额外的深培养皿（Fisher Scientific则），直到它是半满。酷冰，确保板是水平。 </li><li>菜一旦冷却，倒入15毫升的2％琼脂糖顶层。喷一个鸡蛋从一个喷雾瓶的无菌水注入沟槽的塑料模具（自适应科学工具）。小心地将模具槽侧成热的琼脂糖。当琼脂糖冷却后，用金属扁铲（VWR科学）游离阿加模具上升。慢慢地消除从琼脂糖电网。封口膜裹胚胎注射菜（VWR科学）和存储倒在4°C </li><li>对于鱼类幼体注射，准备了2％琼脂糖溶液。倒入一个标准大小的培养皿（VWR科学）解决方案，并拨出，直到它凝固。保鲜膜封口膜和商店在幼虫注射菜倒在4°C </li></ol> 2。真菌培养制备 实验时间:**（30分钟） 难度:** <ol><li>准备酵母提取物，蛋白胨葡萄糖（YPD）琼脂平板:10克/升的酵母提取物，20克/升蛋白胨20克/升，葡萄糖，20克/升的琼脂和釜。为准备的YPD液体10克/升的酵母提取物，20克/升蛋白胨20克/升葡萄糖121和高压灭菌20-30分钟°C。 </li><li>冷冻鱼感染前两天准备的条纹板白念珠菌文化上的YPD琼脂股票获得单菌落。 </li><li>孵育37℃过夜。 </li><li> 5毫升的YPD肉汤放入16 x 150毫米文化管（VWR科学）。 </li><li>鱼感染前一天挑上用木榫（VWR科学）的YPD琼脂1小殖民地。把文化管和漩涡，周围重新挂起殖民地的YPD液体棒。 </li><li>生长在37°C配备一台14英寸的试管轮（新不伦瑞克省科学）上的TC-7组织文化辊鼓过夜。 </li><li>第二天，降速在14000xĞ为1.7毫升管（爱思进）1分钟1毫升的文化。取出上清液，残留液体中悬浮颗粒由涡旋（VWR科学）。 </li><li>加入1 mL 1X的磷酸盐缓冲液（VWR科学）和旋转如前所述。 </li><li>重复PBS洗3次。 </li><li>在1X PBS稀释1:100稀释液（10μL到990μL1X P级水洗白念珠菌BS） </li><li>在血球（VWR科学）计数。 </li><li>稀释至10 7细胞/ ml。 </li></ol> 3。斑马鱼感染 实验时间:****（1-3小时） 难度:**** <ol><li>根据第5条和蛋水店，60 mg / L的无菌去离子水瞬间海洋盐（Fisher Scientific则）收集胚胎。 </li><li>使用，如奥林巴斯SZ61（奥林巴斯），感染当天dechorionate胚胎解剖显微镜。使用杜蒙Dumoxel镊子（VWR科学）拉，就像打开一包薯条，或轻轻捅到闭合位置的绒毛镊子，然后慢慢地打开它们的绒毛27分开。鱼应该弹出出的绒毛。 </li><li>旋流超深培养皿盖子，将鱼入盘的中心。鱼转移到盖子的菜。卸下鸡蛋，水和与新媒体取代。添加鱼类媒体。 </li><li>温暖的幼虫在孵化器注射菜在28°C。准备tricaine甲烷磺酸（西方化学公司）稀释在200微克/毫升麻醉鱼。 </li><li>计算出所需数量的幼虫感染（20-50鱼）。把鱼在tricaine甲烷磺酸盐溶液，等待1-2分钟，直到他们停止移动。 </li><li>打开移动电话伙伴关系-3注射装置（应用科学仪器）。确保压力开关是"脉冲"和"脉冲宽度"设置3 PSI背压机组9。氮气罐阀门打开，直到注射单元上的压力读数为30 PSI。 </li><li> 5μL涡旋念珠菌，1×10 7细胞/毫升的浓度，装入一把拉住微量28。将微量的微量持有人（应用科学仪器）。装满水的超深空培养皿。移动，直到尖端的微量我只是在视图通过解剖显微镜和使用，杜蒙Dumoxel镊子（VWR科学）夹针拔出吸管尖3毫米接触水面。 </li><li>按下脚踏开关（应用科学仪器）来验证针已被截断。你应该看到的液体分散，如果你是成功的。液体丸的直径应不超过整洁25斑马鱼幼虫的瞳孔直径（0.21毫米，产生球体体积的4.9 NL）大。调整的压力，相应地，如果液体丸是太大或太小。 </li><li>麻醉在tricaine甲烷磺酸的鱼（200微克/毫升）。一旦鱼已停止移动，漩涡，直至鱼在中心与盖菜。收集50个鱼用移液管（Fisher Scientific则）。点击移液器解决对尖鱼的一面。到幼虫琼脂糖菜用尽可能少的液体轻轻吸取鱼。 </li><li>线鱼，具有光滑的玻璃棒（小心不粉碎他们！）。吸菜可能关闭的液体。使用KimWipe灯芯了任何水分。 </li><li>直到你得到一个好鱼和针同时认为，放置在显微镜下的仔鱼琼脂糖菜。迈向鱼的玻璃针。在鱼和针定位针对鱼变焦。小心移动到耳泡27，29（EAR）的第一条鱼的玻璃针。你就会知道，一旦你已经搬进针鱼，因为当你把脚踏开关（应用科学仪器），后脑心室将解除略有上升。 </li><li>按下脚踏开关（应用科学仪器），并观看鱼的后脑脑室27内液体分散。收回针从鱼和移动到下一个鱼。重复步骤，直到您已注入盘上所有的鱼。删除任何D的EAD鱼和注入更换，以保持正确的数量（50鱼）。对于每个组50鱼注入了新的显微注射针必须做好准备，或三白色念珠菌在平息针的底部和木屐，或引发的浓度注射。 </li><li>洗净的菜鱼钓鱼菜和干净的超深Petri含有60毫升水蛋菜蛋水喷洒到鱼。重要的是不要离开琼脂糖鱼长于15-20分钟，或他们会干死。 </li><li>重复直到完成整个注塑过程。不要忘了PBS和非致病性注入控制。 </li><li>当完成后，保持在28鱼°C。 </li><li>关闭氮气罐阀门。注射装置的开关从"脉冲"移动"连续"，以纾缓在油箱管路压力，压力应该下降到零，在这一点上。注射装置切换回"把脉"。注射装置关闭和清理工作区。 </li></OL&gt; 4。准备成像鱼 实验时间:**（30分钟） 难度:** <ol><li>前（200微克/毫升）准备tricaine甲烷磺酸的解决方案。走出被感染的鱼，转移到tricaine。 </li><li>准备鸡蛋水47.9 mL 0.4％的低熔点琼脂糖（VWR科学）。通过微波加热解决方案。冷却到37°C和附加tricaine甲烷磺酸盐（200微克/毫升）的混合物</li><li>一旦鱼被固定在tricaine甲烷磺酸，吸管到培养皿中含有0.4％的低熔点琼脂糖（VWR科学）（VWR科学）鱼个体。 </li><li>其次，从低熔点琼脂糖移动到单井成像（MatTek公司）的玻璃底菜的鱼。使用尽可能使鱼平放在盘子的底部在于尽可能少的低熔点琼脂糖。仅使用足够的tricaine琼脂糖填补玻璃底菜NER界。 </li></ol> 5。朱庇特议定书有关的修改微电极用于显微注射 实验时间:*（10-15分钟） 难度:* <ol><li>拉中空玻璃棒BF120-69-10（萨特仪器）使用火焰布朗微管普勒的P-97型（萨特仪器）根据元等30。选择具备下列条件的热量= 470，速度= 120，时间= 200计划＃7。由此产生的针是8毫米，从伞给小费，一旦夹在3毫米以上的小费，它有一个直径约为10微米。 </li><li>装入括号玻璃微。选择"拉"。加热丝将按照程序参数和玻璃棒将分成两个拉微电极针加热。在吸管储物盒（Sutt商店微电极呃文书）。 </li></ol>胚胎收集，吗啉注射液和维护 实验时间:***（1-2小时） 难度:*** <ol start="3"><li>根据Rosen 等31的方法，收集胚胎。使用塑料筛（诺士洁具）收集鸡蛋从产卵池（水生境）。举行筛倒挂一个额外的深培养皿和冲洗水箱，收集鸡蛋。 </li><li>吗啉的制备，添加300μL无菌水300纳摩尔啉（GeneTools，有限责任公司）的。这给出了一个1.0毫米的股票。 </li><li>验证使用的NanoDrop（Thermo Scientific的）吗啉的浓度。选择"核酸和改变的样本类型为"其他"，波长为265。输入常数乘以吗啉1000分子量absorptivit分为Ÿ系数吗啉代寡核苷酸的属性表中列出。 </li><li>空白的NanoDrop 2μL，0.1 N的盐酸。稀释5μL吗啉溶液将达到0.1 N盐酸（20倍稀释）95μL。将2μL稀释的NanoDrop基座和点击的措施。乘以稀释倍数（20）的浓度。这给出了一个工作啉浓度。计算毫摩尔浓度的划分由获得分子量的吗啉的浓度。 </li><li>准备工作的Danieau啉在缓冲区（氯化钠58毫米，0.7毫米氯化钾， 硫酸镁 0.4毫米，0.6毫米的Ca（NO 3）2，5.0毫米肝素钠，pH值7.6）和0.01％酚红（VWR科学）股票。 </li><li>啉注射工作，按照协议，根据Rosen 等31元等30。冲洗啉注射液的菜肴，在28°C，15分钟，鸡蛋，水和温暖。从注射菜肴和线一个-C水成菜用移液管注射（从第1条编制）的凹槽的ELL阶段的胚胎。每块板将容纳大约250个鸡蛋。 1-2细胞阶段的胚胎注入啉。鸡蛋将保持在15分钟27的单细胞阶段。 </li><li>在60毫升的额外深培养皿中蛋水冲洗注入蛋与蛋水结束时（60 mg / L的即时海洋盐无菌去离子水）和地点。可以重复使用吗啉注射菜。蛋水冲洗结束时，并储存在4°C倒置</li><li>对于存储添加60毫升水蛋抛出胚胎。算出来成单独的超深培养皿110胚胎蛋水60毫升，用移液管。添加0.00003％亚甲蓝，以防止微生物的生长和孵化的胚胎，在28°C。 </li><li>要加快胚胎的发育，保持在33°C 24小时和阶段的胚胎菜肴根据金梅尔等27，让胚胎发展到头部主干角度75°（PRIM 25阶段27）。 </li><li>幼虫感染工作，保持在28°C 与 C注射后的胚胎白色念珠菌。 </li><li>每天60毫升新鲜鸡蛋水代替胚胎菜肴。 </li></ol>成像 实验时间:*****（1-5小时） 难度:*** <ol start="14"><li>准备在低熔点琼脂糖（4.2节），如前面所述，在玻璃底部的成像菜（MatTek公司）tricaine鱼。奥林巴斯IX-81（奥林巴斯）倒置显微镜放在菜。微分干涉对比（DIC）的光下，带来4倍放大倍率下的鱼成焦点。图像可以被捕获的4倍，20倍，并在DIC，TRITC（四甲基若丹明异硫氰酸）和FITC（异硫氰酸荧光素）的过滤器设置，40倍的放大倍率。 </li><li>使用FV-1奥林巴斯IX-81倒置显微镜000激光扫描共聚焦系统有贺等32。进行40倍放大倍率的时间课程设置Z轴堆叠1-1.5微米片每隔一小时感染。 </li><li> 2个小时或更长时间当然成像，放置在顶部的在成像菜鱼每隔2-3小时的低熔点琼脂糖层。这可以防止干燥和粉碎的鱼，而它正在成像琼脂糖。对于一个小的鱼数量的漫长时间的课程，使用了激烈的阶段（Bioptechs公司）保持在29°C感染。 </li></ol> 6。代表结果例如一个成功的后脑脑室C.白色念珠菌感染，在斑马鱼幼虫在5小时后感染（HPI）和24 HPI所示（ 图1）。巨噬细胞样细胞，吞噬C。白念珠菌被认为是在5 HPI的后脑心室。 24 HPI，C.白色念珠菌是内巨噬细胞样细胞S在背尾组织的播散性念珠菌指示。这种感染的结果是高度依赖10-15 C.酵母形式的准确注射后白念珠菌到后脑心室。被感染的鱼立即注射后的筛选，可以确保这一点。 <img alt="图1" src="/files/ftp_upload/4051/4051fig1.jpg" /> 图1转基因FLI1:绿色荧光蛋白22，氟化钙，yCherry 白念珠菌和共聚焦显微镜成像intravitally 33幼虫感染。 （AC）5小时后感染（一）-EGFP表达巨噬细胞样细胞在感染部位（后脑心室）比例尺= 100微米的感染幼虫。 （B和C）高倍率图像相同的鱼，显示C。白色念珠菌在吞噬。比例尺= 100微米和10微米为C.（DF）的氟化钙，yCherry C.传播与念珠菌感染后24小时（D）与被感染的幼虫白念珠菌内EGFP的MAC在背尾组织的巨噬细胞的细胞。比例尺= 100微米。 （E和F）高倍率图像相同的鱼，显示C。白色念珠菌在尾部组织。比例尺= 100微米。

没有利益冲突的声明。

这里的斑马鱼显微注射的方法不同于，Gutzman 等。在这里34我们展示通过注射到36至48 HPF幼虫的后脑心室耳泡。我们所描述的方法可以减少组织损伤后脑心室10-15酵母一致的注入。该协议产生的最初24 HPI（ 图1）造成重大杀伤力/ 5发病，整个身体蔓延的局部感染。后脑脑室不完全封闭，直到48 HPF 27，29。在此发展的巨噬细胞和中性粒细胞的早期迁移到感染?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>产卵箱</td><td>水生栖息地</td><td> 2L </td><td></td></tr><tr><td> 1.7 mL管</td><td>爱思进</td><td> MCT-175-C的</td><td></td></tr><tr><td>即时海洋</td><td> Fisher Scientific则</td><td> S17957C </td><td></td></tr><tr><td>超深培养皿</td><td> Fisher Scientific则</td><td> 08-757-11Z </td><td></td></tr><tr><td>标准的培养皿</td><td>厂商VWR科学</td><td> 25384-302 </td><td></td></tr><tr><td>转移移液器</td><td> Fisher Scientific则</td><td> 13-711-7M </td><td></td></tr><tr><td>酵母提取物</td><td>厂商VWR。科学家FIC </td><td> 90000-726 </td><td></td></tr><tr><td>蛋白胨</td><td>厂商VWR科学</td><td> 90000-264 </td><td></td></tr><tr><td>葡萄糖</td><td> Fisher Scientific则</td><td> D16-1 </td><td></td></tr><tr><td>洋菜</td><td>厂商VWR科学</td><td> 90000-760 </td><td></td></tr><tr><td>一次性血球</td><td>厂商VWR科学</td><td> 82030-468 </td><td></td></tr><tr><td>磷酸盐缓冲液</td><td>厂商VWR科学</td><td> 12001-986 </td><td></td></tr><tr><td>杜蒙Dumoxel镊子</td><td>厂商VWR科学</td><td> 100501-806 </td><td></td></tr><tr><td>木销钉</td><td>厂商VWR科学</td><td> 10805-018 </td><td></td></tr><tr><td> KimWipes </td><td>厂商VWR科学</td><td> 300053-964 </td><td></td></tr><tr><td>低熔琼脂糖</td><td>厂商VWR科学</td><td> 12001-722 </td><td></td></tr><tr><td>琼脂糖为注射菜</td><td>厂商VWR科学</td><td> 12002-102 </td><td></td></tr><tr><td>火焰山布朗微管普勒</td><td>萨特仪器</td><td>的P-97 </td><td></td></tr><tr><td>中空玻璃棒</td><td>萨特仪器</td><td> BF120-69-10 </td><td>对于光滑的玻璃玻璃棒本生灯加热</td></tr><tr><td>吸取存储盒</td><td>萨特仪器</td><td> BX10 </td><td></td></tr><tr><td> MPPI-3喷射系统</td><td>应用科学仪器</td><td>移动电话伙伴关系-3 </td><td></td></tr><tr><td>背压单位</td><td>应用科学仪器</td><td>边境巡逻队</td><td></td></tr><tr><td>微量持有人套件</td><td> APPLIED科学仪器</td><td> MPIP </td><td></td></tr><tr><td>脚踏开关</td><td>应用科学仪器</td><td>搅拌摩擦焊</td><td></td></tr><tr><td>微操作机器人</td><td>应用科学仪器</td><td> MM33 </td><td></td></tr><tr><td>磁性底座</td><td>应用科学仪器</td><td>磁性底座</td><td></td></tr><tr><td> tricaine甲烷磺酸盐</td><td>西方化工公司</td><td> MS-222 </td><td></td></tr><tr><td>解剖范围</td><td>奥林巴斯</td><td> SZ61顶端SZX-ILLB2-100基地</td><td></td></tr><tr><td>共聚焦显微镜</td><td>奥林巴斯</td><td> IX-81 FV-1000激光扫描共聚焦系统</td><td></td></tr><tr><td> TC-7组织文化与14英寸试管轮辊筒</td><td>新不伦瑞克省科技</td><td> TC-7 </td><td></td></tr><tr><td>成像菜</td><td> MatTek公司</td><td> P24G-1.0-10 - F </td><td></td></tr><tr><td>用于装载针枪头</td><td> Eppendorf公司</td><td> 930001007 </td><td></td></tr><tr><td>板浇筑电网</td><td>自适应科学工具</td><td> TU-1 </td><td></td></tr><tr><td>加热阶段</td><td> bioptechs公司</td><td>三角洲T-5 </td><td></td></tr><tr><td>扁铲</td><td>厂商VWR科学</td><td> 82027-486 </td><td></td></tr><tr><td>塑料分子筛</td><td>制品的诺士佛在线</td><td> 12厘米</td><td></td></tr><tr><td>封口膜</td><td>厂商VWR科学</td><td> 52858-000 </td><td></td></tr><tr><td>涡精灵</td><td>厂商VWR科学</td><td> 14216-184 </td><td></td></tr><tr><td> 16 x 150毫米的文化管</td><td>厂商VWR科学</td><td> 60825-435 </td><td></td></tr><tr><td>的NanoDrop </td><td> Thermo Scientific的</td><td> ND折价2000 </td><td></td></tr><tr><td>酚红</td><td>厂商VWR科学</td><td> 97062-478 </td><td></td></tr><tr><td>盐酸</td><td>厂商VWR科学</td><td> 87003-216 </td><td></td></tr><tr><td>氯化钠</td><td>厂商VWR科学</td><td> BDH4534-500GP </td><td></td></tr><tr><td>氯化钾</td><td>厂商VWR科学</td><td> BDH4532-500GP </td><td></td></tr><tr><td> 硫酸镁 </td><td>厂商VWR科学</td><td> BDH0246-500GP </td><td></td></tr><tr><td>的Ca（NO 3）2 </td><td>厂商VWR科学</td><td> BDH0226-500GP </td><td></td></tr><tr><td>肝素钠</td><td>厂商VWR科学</td><td> BDH4520-500GP </td><td></td></tR&gt; <tr><td>吗啉代</td><td> GeneTools，LLC </td><td></td><td></td></tr></tbody></table>

non-invasive imaging of disseminated candidiasis in zebrafish larvae

由病原体白念珠菌引起的播散性念珠菌是临床上重要的问题，在住院的个人和30到40％的占死亡率6。系统性念珠菌通常是由先天免疫控制，并与先天免疫细胞成分如巨噬细胞NADPH氧化酶的遗传缺陷的人更易患念珠菌7-9。很少有人知道关于C 的动态白念珠菌与先天免疫细胞在体内的相互作用。在体外研究中广泛建立了外部主机C.白念珠菌的巨噬细胞内发芽，迅速 ​​摧毁了嗜中性粒细胞10-14。 在体外研究中，虽然有用，能不能概括在复杂的体内环境，细胞因子水平，细胞外基质的附件，以及10间的接触，其中包括随时间变化的动态， 15-18 </sup&gt;。为了探讨这些因素在宿主 - 病原体相互作用的贡献，关键是找到一个模式生物，在现场的完整主机可视化的非侵入性感染的这些方面。 斑马鱼幼虫提供了一个独特的和多功能的脊椎动物宿主感染的研究。发展的斑马鱼幼虫的前30天，有先天免疫防御2，19-21，简化，如播散性念珠菌是高度依赖先天免疫疾病的研究。小斑马鱼幼虫的大小和透明度，使在细胞水平上的主机和病原体感染的动态影像。与先天免疫细胞荧光的转基因幼虫可用于识别特定的细胞类型涉及在感染22-24。修饰的反义寡核苷酸（吗啉代）可以用来击倒各种免疫成分，如巨噬细胞NADPH氧化酶研究在回应funga的变化L型感染5。除了使用小低脊椎动物的道德和实用性强等优点，斑马鱼幼虫提供了独特的图像之间的病原体和宿主在intravitally颜色的酣战的可能性。 斑马鱼已被用于模型感染人类致病细菌的数量，并一直在我们理解分枝杆菌感染，25器乐重大进展。然而，最近才具有更大的病原体，如被用于真菌感染的幼虫5，23，26，迄今没有发生过感染的方法进行了详细的视觉描述。在这里，我们提出后脑普里姆25斑马鱼心室显微注射，包括我们以前的协议的修改我们的技术。 在体外研究中我们发现真菌感染的幼虫斑马鱼模型偏离和加强需要检查主机的病原体INTERAction培养皿5简化了系统的主机，而不是在复杂的环境中。

Watch this Scientific Journal Video about Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae at JoVE.com

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

斑马鱼的快速发展，小尺寸和透明度是先天感染的免疫控制的研究的巨大优势<sup&gt; 1-4</sup&gt;。在这里，我们证明斑马鱼幼虫病原真菌感染的技术<em&gt;白念珠菌</em&gt;通过显微注射方法最近使用的牵连吞噬细胞NADPH氧化酶活性真菌异形控制<sup&gt; 5</sup&gt;。

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 ...

non-invasive-imaging-of-disseminated-candidiasis-in-zebrafish-larvae

Research

JoVE Journal

Immunology and Infection

14.6K 次观看.  University of Maine. 斑马鱼的快速发展，小尺寸和透明度是先天感染的免疫控制的研究的巨大优势 1-4。在这里，我们证明斑马鱼幼虫病原真菌感染的技术白念珠菌通过显微注射方法最近使用的牵连吞噬细胞NADPH氧化酶活性真菌异形控制 5。

视频: Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

白细胞的归位和迁移的非侵入性的成像在体内</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

科研

免疫与感染

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

非侵入性光学成像淋巴血管的鼠标

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Neuropathogenic的非侵入型<em&gt;大肠杆菌</em&gt;感染乳鼠

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and pneumothorax from pleural disruption can result in a significant mortality rate. This protocol is a simple method of rat thymectomy that utilizes a mini-sternotomy and endotracheal intubation. Intubation is accomplished with a non-invasive and easily reproducible method and allows for positive pressure ventilation to prevent pneumothorax and a controlled airway that allows sufficient time for careful thymus dissection to minimize pleural disruption. A 1.5 cm sternal incision decreases contact with mediastinal vessels and pleura, while still providing full visualization of the thymus. Following exposure of the mediastinum, the thymus is removed by blunt dissection under magnification. The pleural space is then sealed by suture closure of the pre-tracheal muscles followed by the application of surgical glue. The thorax is then closed by suture closure of the sternum, followed by suture closure of the skin. All thymectomies were complete as evidenced by immunohistochemical (IHC) staining of mediastinal tissue, and absence of na&#239;ve T-cells by flow cytometry, and the procedure had a 96% survival rate. This method is suitable when complete thymectomy with minimal complications is desired for further immunological studies in athymic adult rats.

Rodent thymectomy is a valuable technique in immunological research. Here, a protocol for complete thymectomy in adult rats using a mini-sternotomy along with non-invasive intubation and positive pressure ventilation to minimize perioperative morbidity and mortality is described.

完整的胸腺切除术的成年大鼠非侵入气管插管

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and ...

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their intercommunication, are paramount for the protection against infections. The interactions of epithelial and innate immune cells with a pathogen are best investigated in vivo, where complex behavior unfolds over time and space. However, existing models do not allow for easy spatio-temporal imaging of the battle with pathogens at the mucosal level.
The model developed here creates a mucosal infection by direct injection of the fungal pathogen, Candida albicans, into the swimbladder of juvenile zebrafish. The resulting infection enables high-resolution imaging of epithelial and innate immune cell behavior throughout the development of mucosal disease. The versatility of this method allows for interrogation of the host to probe the detailed sequence of immune events leading to phagocyte recruitment and to examine the roles of particular cell types and molecular pathways in protection. In addition, the behavior of the pathogen as a function of immune attack can be imaged simultaneously by using fluorescent protein-expressing C. albicans. Increased spatial resolution of the host-pathogen interaction is also possible using the described rapid swimbladder dissection technique.
The mucosal infection model described here is straightforward and highly reproducible, making it a valuable tool for the study of mucosal candidiasis. This system may also be broadly translatable to other mucosal pathogens such as mycobacterial, bacterial or viral microbes that normally infect through epithelial surfaces.

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

造型粘膜念珠菌在斑马鱼幼虫通过鳔注射

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

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

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

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 ...

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 ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

在小鼠实验为细菌性肺炎的诱导和表型非侵入性和技术上非密集型方法

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.
Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.

无创<em&gt;体内</em炎性MMP活性的使用可激活荧光成像剂&gt;荧光光学成像

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is that they may induce traumatic lesions on the surface of the eye, which can progress to scarring, eyelid deformity, limbal stem cell deficiency and in some cases, cause great discomfort to the subject. To avoid these clinical problems, impression cytology (IC) was developed to diagnose dry eye disease and later neoplasia, atopic disease, vernal keratoconjunctivitis and keratoconjunctivitis sicca. Typically, clinicians manually cut filter papers into required shapes and apply these to the ocular surface. Here, we describe how to perform IC using a commercially available medical device. This technique is explained here followed by immunophenotyping by flow cytometry. This technique requires less manual handling and causes less injury to the ocular surface.

The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva.

从结膜分离和表型细胞的非侵入性方式

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is ...