这项工作是支持授权号码R01AI055001和R01AI082127从NIAID的自然资源管理和SREB论文奖学金奖JLB。

1。荧光标记的葡聚糖和金思朗我isolectin乙面部静脉注射标签胚胎血管<ol><li>准备FITC -葡聚糖（50ug/mL）在磷酸盐缓冲液（PBS）或金思朗1 - isolectin乙4（20ug/200uL）在1.5ml的Eppendorf管中，并在PBS温暖至37 ° C 。股票1.25MM快速绿/ PBS 100uL FITC -葡聚糖溶液（总体积毫升）和股票1.25MM快速180uL绿/ PBS金思朗1 - isolectin乙4（总体积200uL），这样的解决方案是明显的蓝色。 </li><li>解剖E14.5 - E18.5胚胎和卵黄囊在一起，留下的尿囊柄（脐动脉和静脉）不变。 </li><li>胚胎转移到一个新的皮特里盘（60 × 15毫米），他们沉浸在PBS在室温下。 </li><li>胚胎的位置，以提供的头部/面部矢状。使用微解剖钳轻轻抓住头胚胎。 </li><li>使用30G针头，注入50uL FITC -葡聚糖（50ug/mL）或金思朗1 -到面部静脉针指 ​​向的后脑勺isolectin乙4（20ug 200uL PBS）。 </li><li>当染料是在脐静脉可见，去掉针头和独立的胚胎从尿囊柄（脐动脉和静脉）。 </li><li>注射之后，让胚胎在室温下PBS为2-3分钟，使染料整个胚胎循环。 </li></ol> 2。全安装皮肤血管分析<ol><li>使染料流通整个胚胎后，取下皮肤样本地区的四肢，背部，和胃，等8,9。 </li><li>在冷PBS洗皮肤样本，并在4％PFA / PBS固定为2小时8,9。 10分钟，每个4ML明确小瓶用2ml冷PBS洗3次。 </li><li>放在皮肤样本在显微镜幻灯片，并添加100μL安装媒体每张幻灯片和一个玻璃盖。 </li><li>允许的幻灯片，以干在黑暗的存储区域。 </li><li>继续执行步骤2的图像采集“部分。 </li></ol> 3。胸腺及心脏血管，血管周围细胞和冰冻切片的多色标签（继续从第1款，第7步） <ol><li> “闪光冻结”整个胚胎在液氮中。胚胎可以储存于-80 ° C，直到分析。 </li><li>另外，解剖出胸腺，在4 °彗星PBS冲洗，并在2小时修复液2ml 4％多聚甲醛（PFA）/ PBS。洗3次，地点在华侨城thymi冷PBS，并冻结和存储10分钟，直到使用在-80 ° C </li><li> cryosectioning，传播“块”一节OCT和安装的胚胎或切片解剖器官/组织。 </li><li>切成10微米厚的冰冻组织和收集的幻灯片。 </li><li>修复部分在5-10分钟丙酮。在寒冷的TBS清洗3次。 </li><li>在室温湿度试验箱在10％驴血清/ TBS座。 </li><li>孵育1初级抗体100μL湿度试验箱在4小时通宵部分℃：在这个例子中，我们使用大鼠抗小鼠CD31（1:100）标签内皮细胞和山羊抗鼠PDGFR -β （1:100）标签血管周围细胞。它是有用的覆盖与单独切割的封口膜条幻灯片，以确保该抗体是统一部分蔓延。 </li><li>初级抗体的潜伏期后，在寒冷的TBS洗节3倍。 100μL适当的二级抗体孵育至少30分钟。 </li><li>在寒冷的TBS清洗3次。加入100μL安装媒体每张幻灯片和一个玻璃盖。 </li><li>允许的幻灯片，以干在黑暗的存储区域。 </li><li>继续执行“图像采集”部分。 </li></ol> 4。 vibratome部分胸腺血管和血管周围细胞的多色标签（继续从第1款，第7步） <ol><li>解剖出胚胎胸腺叶，冷PBS冲洗。 </li><li> 4％PFA / PBS在室温下2小时修复胸腺。 </li><li>在PBS - TRITON X（0.15％）的3倍，10分钟和地方thymi洗净，在一个小塑料盒和淹没在4％的低熔融琼脂糖/ PBS（4℃）。胸腺应与墨盒底部的接触。 </li><li>允许琼脂糖上冰（3-5分钟），以巩固。使用刀片，切断多余的琼脂糖。添加胶水vibratome块，并坚持以块样品。 </li><li>直到样品和刀片都沉浸冷PBS vibratome水浴。 </li><li>设置速度和幅度（高幅度和低适中的速度，是理想的软胸腺部分）。如果部分打破由于多余的搅拌，应减少的幅度。 </li><li>切割50微米的部分。 </li><li>使用画笔，在冷PBS 24孔微孔板收集部分。 </li><li>块切片在500μL10％驴血清PBS - TRITON X为30分钟（0.15％）。 </li><li>孵育部分为8小时初级抗体过夜，如抗CD31和抗- PDGFR -β在有盖的24孔微孔板。 </li><li>洗净比PBS - TRITON X 3次（0.15％）共8个小时，在4 ° C。 </li><li>块切片在10％驴血清PBS - TRITON X为30分钟（0.15％）。 </li><li>孵育4℃，适当的二次抗体的部分为8小时至过夜。 </li><li>洗净比PBS - TRITON X 3次（0.15％）共8个小时，在4 ° C。 </li><li>重新修复的样品在4％PFA / PBS在冰上30分钟。 </li><li>在冰上超过30分钟，洗净PBS - TRITON X 3次（0.15％）。 </li><li>通过分级甲醇/ PBS -海神X系列脱水样本：25％甲醇，50％甲醇，75％甲醇，每一步都在10分钟和100％甲醇。 10分钟后，替换100％，与新鲜甲醇的甲醇和1小时在室温下孵育。 </li><li>在一个玻璃容器中，在1:2的比例混合BABB（苯甲醇苯甲酸苄酯）。为终浓度为50％和50％的BABB甲醇与甲醇结合BABB。孵育BABB样品：甲醇为10-15分钟。 </li><li>转移样品到玻璃容器100％BABB孵育10-15分钟或直到被清除，在室温下。 </li><li>填充新鲜的100％的BABB和转让样品侧面抑郁幻灯片（0.7毫米深度）。 2-3指甲油大衣盖玻璃（1.5号）和密封。允许波兰在室温下硬化钉在黑暗中，然后将其存储样品在4 ° C。 注：幻灯片等，必须完全密封的共聚焦图像采集前。图像应在12-24小时之内，收购在BABB荧光染料褪色。 </li><li>继续执行“图像采集”部分。 </li></ol> 5。图像采集<ol><li>图片10微米的冰冻切片，共聚焦显微镜使用488计划复消色差透镜20X/0.8目标（512 x 512像素） - （FITC-dextran/GSL 1 - isolectin乙4），543 - ，和633纳米的激光线。 </li><li>收购焦Z -整装皮肤和50微米琼脂糖包埋切片使用488计划复消色差透镜10X/0.4目标（512 x 512像素）的部分- （FITC-dextran/GSL 1 - isolectin 乙 4），543 - 633纳米的激光线。串行Z型截面应收集在1微米顺序，每个相应的通道。 </li><li>重建串行Z轴部分使用蔡司Axiovision 4.6或其他图像分析软件。 </li></ol> 6。代表性的成果： 胚胎血管的有效标签是评估在胚胎小鼠的血管缺陷的关键。图1显示了特定的标签E16.5胸腺血管（1A - B）和CD31（1B）的共同标签，除了左，右心室的染色（1E - F），分别。金思朗我isolectin乙4协议是在第1，第3和5中描述的冰冻切片在这些实验中使用。图1C - D E16.5小鼠皮肤的血液血管网全安装的标签，使用在第1，第2条所述的协议，和5所示。 <img alt="图1" src="/files/ftp_upload/3267/3267fig1.jpg" /> 图1传奇。 FITC金思朗我- isolectin乙4到E16.5小鼠胚胎的面部静脉注射 。 E.胚胎心脏E. F. Cryosection（右A. Cryosection胚胎胸腺注射后CD31的共同标签。B.合并isolectin B级4。C.和D.全胚胎皮肤血管注射后安装。心室）F.（左心室）注射后。

<ol>
	<li>Cuddihy, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-mediated+cross-talk+within+the+neonatal+murine+thymus.">VEGF-mediated cross-talk within the neonatal murine thymus.</a> Blood. 113, 2723-2731 (2009).</li><li>Muller, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+targeting+of+VEGF-A+in+thymus+epithelium+disrupts+thymus+blood+vessel+architecture.">Gene targeting of VEGF-A in thymus epithelium disrupts thymus blood vessel architecture.</a> Proc. Natl. Acad. Sci. U. S. A. 102, 10587-10592 (2005).</li><li>Muller, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+origin+of+perivascular+mesenchyme+in+the+adult+thymus.">Neural crest origin of perivascular mesenchyme in the adult thymus.</a> J. Immunol. 180, 5344-5351 (2008).</li><li>Foster, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+neural+crest-derived+cells+in+the+embryonic+and+adult+thymus.">Contribution of neural crest-derived cells in the embryonic and adult thymus.</a> J. Immunol. 180, 3183-3189 (2008).</li><li>Liu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordination+between+CCR7-+and+CCR9-mediated+chemokine+signals+in+prevascular+fetal+thymus+colonization.">Coordination between CCR7- and CCR9-mediated chemokine signals in prevascular fetal thymus colonization.</a> Blood. 108, 2531-2539 (2006).</li><li>Lavine, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor+signals+regulate+a+wave+of+Hedgehog+activation+that+is+essential+for+coronary+vascular+development.">Fibroblast growth factor signals regulate a wave of Hedgehog activation that is essential for coronary vascular development.</a> Genes Dev. 20, 1651-1666 (2006).</li><li>Lavine, K. J., Kovacs, A., Ornitz, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+signaling+is+critical+for+maintenance+of+the+adult+coronary+vasculature+in+mice.">Hedgehog signaling is critical for maintenance of the adult coronary vasculature in mice.</a> J. Clin Invest. 118, 2404-2414 (2008).</li><li>Mukouyama, Y. S., Gerber, H. P., Ferrara, N., Gu, C., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+nerve-derived+VEGF+promotes+arterial+differentiation+via+neuropilin+1-mediated+positive+feedback.">Peripheral nerve-derived VEGF promotes arterial differentiation via neuropilin 1-mediated positive feedback.</a> Development. 132, 941-952 (2005).</li><li>Mukouyama, Y. S., Shin, D., Britsch, S., Taniguchi, M., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+nerves+determine+the+pattern+of+arterial+differentiation+and+blood+vessel+branching+in+the+skin.">Sensory nerves determine the pattern of arterial differentiation and blood vessel branching in the skin.</a> Cell. 109, 693-705 (2002).</li><li>Murphy, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+Notch4+signaling+induces+hallmarks+of+brain+arteriovenous+malformations+in+mice.">Endothelial Notch4 signaling induces hallmarks of brain arteriovenous malformations in mice.</a> Proc. Natl. Acad. Sci. U. S. A. 105, 10901-10906 (2008).</li></ol>

没有利益冲突的声明。

全安装和PECAM - 1（CD31）染色部分标签在胚胎小鼠的血管的常规方法。这些方法需要使用的直接和/或间接免疫荧光和洗涤剂通透小鼠组织。这被证明是一个相当及时处理。在这里，我们有FITC -葡聚糖或isolectin B级4面部静脉注射直接标记的胚胎血管，从而消除了抗体标记的步骤要求。此外，这种方法使血管功能状态的评估，将通过这种方法，而不是由传统抗体染色方法显示“漏水”的表型。...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td> FITC -葡聚糖</td><td>西格玛</td><td> FD150S - 1G </td></tr><tr><td>荧光标记金思朗1 - 4 isolectin乙</td><td>媒介实验室</td><td> FL - 1201 </td></tr><tr><td>快绿</td><td> MP生物医学</td><td> 195178 </td></tr><tr><td>煤灰</td><td> Fluka公司</td><td> 76240 </td></tr><tr><td>胎牛血清</td><td>亚特兰大生物</td><td> S11550 </td></tr><tr><td>最佳切削温度化合物（华侨城</td><td> VWR </td><td> 25608-930 </td></tr><tr><td>丙酮</td><td> JT贝克</td><td> 9006-33 </td></tr><tr><td>驴血清</td><td>杰克逊</td><td> 017-000-121 </td></tr><tr><td>大鼠抗小鼠CD31， </td><td> BD Pharmingen公司</td><td> 558736 </td></tr><tr><td>羊抗鼠PDGFR -β </td><td> R＆D系统</td><td> AF1042 </td></tr><tr><td>驴抗大鼠CD31的Alexa的647（Invitrogen公司） </td><td> Biolegend </td><td> 102516 </td></tr><tr><td>驴抗山羊Alexa的594（Invitrogen公司） </td><td> Invitrogen公司</td><td> A11058 </td></tr><tr><td> TRITON X -100 </td><td> Sigma - Aldrich公司</td><td> X - 100 </td></tr><tr><td>低熔点琼脂糖/ PBS </td><td> Sigma - Aldrich公司</td><td> A9414 - 25G </td></tr><tr><td>甲醇</td><td> Fisher Scientific则</td><td> A413 - 4 </td></tr><tr><td>苯甲醇</td><td>阿库罗斯福冈科学</td><td> 148390010 </td></tr><tr><td>苯甲酸苄酯</td><td>阿库罗斯福冈科学</td><td> 105860010 </td></tr><tr><td>抑郁症的幻灯片</td><td> Fisher Scientific则</td><td> S175201 </td></tr><tr><td> Fluorogel </td><td>电子显微镜科学</td><td> 17985-10 </td></tr><tr><td>玻璃盖（22X22）-1.5 </td><td> Thermo Scientific的</td><td> 152222 </td></tr><tr><td>蔡司LSM 510 META共聚焦显微镜</td><td>蔡司</td><td></td></tr><tr><td>微解剖钳</td><td> Roboz </td><td> RS - 5135 </td></tr><tr><td>封口膜号OM992 </td><td> Fisher Scientific则</td><td> 13-374-16 </td></tr><tr><td> 12和24孔板</td><td>长荣科学</td><td> 222 - 8044 - 01F </td></tr><tr><td> Superfrost / PLUS显微镜载玻片</td><td> Fisher Scientific则</td><td> 12-550-15 </td></tr><tr><td> 4ML明确小瓶</td><td>国家科学技术进步奖</td><td> B7800 - 2 </td></tr></tbody></table>

a method for labeling vasculature in embryonic mice

功能的血管网络的建立是一个器官形成的重要组成部分，是最佳的器官功能要求。例如，在胸腺适当的血管形成和图案是胸腺细胞进入到器官和成熟的T细胞向周边出口所必需的。在胸腺血管的空间安排，是依赖于从局部微环境的信号，即胸腺上皮细胞（TEC）。最近的一些报告表明，这些信号结果在胸腺血管缺陷1,2中断。以前的研究已经描述技术用于标签的新生儿和成人的胸腺血管 1,2 。我们在这里展示一个标签血管在胚胎胸腺技术。这种方法结合使用FITC -葡聚糖或加纳（Bandeiraea）Simplicifolia凝集素我（1 - GSL isolectin乙4）面部静脉注射和CD31抗体染色，以确定胸腺的血管结构和PDGFR -β标签胸腺血管周围间质 3-5 。还提供了使用冰冻切片或vibratome部分选项。该协议可用于识别胸腺血管的缺陷，这是定义在胸腺血管形成的TEC -派生分子的角色的关键。作为方法的标签，整个血管，它也可以被用来分析多个器官和整个组织，包括皮肤和心脏 6-10胚胎的血管网络。

Watch this Scientific Journal Video about A Method for Labeling Vasculature in Embryonic Mice at JoVE.com

A Method for Labeling Vasculature in Embryonic Mice

本文介绍了标签胚胎皮肤和胸腺血管的方法。

标签胚胎小鼠血管的方法

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

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Research

JoVE Journal

Immunology and Infection

15.5K 次观看.  University of Georgia. 本文介绍了标签胚胎皮肤和胸腺血管的方法。

视频: A Method for Labeling Vasculature in Embryonic Mice

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

科研

免疫与感染

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

V3的HIV - 1取向的基因型推理使用人口为基础的测序

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

可视登革病毒通过的Alexa Fluor标签

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

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 ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

4D多模态成像<em&gt;柠檬酸rodentium</em在小鼠感染

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

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 ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

诱导麻痹及视觉系统损伤小鼠 T 细胞特异的视神经脊髓炎抗原水通道蛋白-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

小鼠涎腺功能的测定方法

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

Deep Vein Thrombosis Induced by Stasis in Mice Monitored by High Frequency Ultrasonography

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death through pulmonary embolism or results in the post-thrombotic syndrome, characterized by chronic leg pain, swelling, and ulceration, or in chronic pulmonary hypertension resulting in significant chronic respiratory compromise. This is the most common cardiovascular disease after myocardial infarction and ischemic stroke and is a clinical challenge for all medical disciplines, as it can complicate the course of other disorders such as cancer, systemic disease, surgery, and major trauma.
Experimental models are necessary to study these mechanisms. The stasis model induces consistent thrombus size and a quantifiable amount of thrombus. However, it is necessary to systematically ligate side branches of the inferior vena cava to avoid variability in thrombus sizes and any erroneous data interpretation. We have developed a non-invasive technique to measure thrombus size using ultrasonography. Using this technique, we can assess thrombus development and resolution over time in the same animal. This approach limits the number of mice required for quantification of venous thrombosis consistent with the principle of replacement, reduction, and refinement of animals in research. We have demonstrated that thrombus weight and histological analysis of thrombus size correlate with measurement obtained with ultrasonography. Therefore, the current study describes how to induce deep vein thrombosis in mice using the inferior vena cava stasis model and how to monitor it using high frequency ultrasound.

The present protocol describes the steps to obtain venous thrombosis using a stasis model. In addition, we are using a non-invasive method to measure thrombus formation and resolution over time.

高频超声监测小鼠瘀血致深静脉血栓形成

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death ...

A Silver Nanoparticle Method for Ameliorating Biliary Atresia Syndrome in Mice

Biliary atresia (BA) is a severe type of cholangitis with high mortality in children of which the etiology is still not fully understood. Viral infections may be one possible cause. The typical animal model used for studying BA is established by inoculating a neonatal mouse with a rhesus rotavirus. Silver nanoparticles have been shown to exert antibacterial and antiviral effects; their function in the BA mouse model is evaluated in this study. Currently, in BA animal experiments, the methods used to improve the symptoms of BA mice are generally symptomatic treatments given via food or other drugs. The aim of this study is to demonstrate a new method for ameliorating BA syndrome in mice by the intraperitoneal injection of silver nanoparticles and to provide detailed methods for preparing the silver nanoparticle gel formulation. This method is simple and widely applicable and can be used to research the mechanism of BA, as well as in clinical treatments. Based on the BA mouse model, when the mice exhibit jaundice, the prepared silver nanoparticle gel is injected intraperitoneally to the surface of the lower liver. The survival status is observed, and biochemical indicators and liver histopathology are examined. This method allows a more intuitive understanding of both the establishment of the BA model and novel BA treatments.

This article describes in detail a method based on silver nanoparticles for ameliorating biliary atresia syndrome in an experimental biliary atresia mouse model. A solid understanding of the reagent preparation process and the neonatal mouse injection technique will help familiarize researchers with the method used in neonatal mouse model studies.

银纳米粒子法改善小鼠胆道闭锁综合征

Biliary atresia (BA) is a severe type of cholangitis with high mortality in children of which the etiology is still not fully understood. Viral infections ...

Methods Article

标签胚胎小鼠血管的方法