这项工作是由在加拿大卫生研究院，加拿大不列颠哥伦比亚大学中心的创新，血液研究和北卑诗大学基金会。

1。试剂准备<ol><li>在整个实验中使用的试剂应在开始手术协议前准备。请注意，所有的解决方案是采用无菌技术。 </li><li>生理盐水溶液（PSS）是最好准备两部分组成：基本盐溶液和碳酸氢钠溶液。准备在这两种解决方案20X浓度和储存在4 ° C。碳酸氢钠在20X 360.0mM碳酸氢钠（84.01g/mol）和基本20X ​​浓度盐溶液94.0mM 2638.0mM氯化钠（58.44g/mol），氯化钾（74.56g/mol），40.0mM 氯化钙 •2H 2为O（147.02g/mol），并23.4mM硫酸镁4•7H 2 O（246.48g/mol）。应准备在DH 2 O和化学品的解决方案应补充一次，搅拌，直到解决方案之前加入未来的化学品</li><li>准备的PSS 2L等量的碳酸氢钠和基本盐溶液稀释，稀释至1X浓度（的PSS 2L每个碳酸氢钠和基本盐溶液稀释100毫升卫生署2 </li><li>置于2L容量瓶在37℃水浴中含有PSS和PSS。 1小时前平衡的解决方案，以外科手术开始，用5％的CO 2 /平衡氮气。 </li><li>血管活性化学物质（如苯肾上腺素，乙酰胆碱，硝普钠）是最好的准备1M浓度在dH2O和100μL分装储存于-20 ° C </li></ol> 2。动物的制备<ol><li>确定鼠标的重量和管理的初始剂量的戊巴比妥钠腹腔注射每千克90毫克。整个过程中，鼠标不断监测麻醉通过脚趾捏和呼吸频率的仔细检查飞机。管理附加注射戊巴比妥钠20mg/kg维持麻醉平面的需要。手术的麻醉平面维持在联邦和机构法规的规定，动物护理和使用北卑诗大学（ACUC）委员会批准的协议动物保健概述。 </li><li>删除从腹部和侧面/臀面积由剃须刀的头发。删除剩余的酒精棉签松散的头发，拍着用胶带面积。这是足够的，因为这样的实验过程是终端。 </li><li>在仰卧位放置在一个明确的手术板鼠标和附加录音每个脚垫板鼠标板。保持体温是通过外部加热灯，并通过直肠温度计监测，以确保低温麻醉剂不会发生。 </li></ol> 3。外科手术<ol><li>确保在整个手术过程中暴露的组织始终保持平衡回暖的PSS液灌流。同样重要的是永远不会与血管手术器械的利益​​接触或放置在他们用力过猛，通过操纵他们周围的脂肪组织和结缔组织的伤害船只。 </li><li>将鼠标下的解剖范围。沿着腹面中线切开腹腔，并收回对鼠标的脊柱皮肤。 </li><li>一旦皮肤回缩，使LN是很容易看到，针到一个透明Sylgard 184底座的皮肤。 </li><li>删除的一层薄的结缔组织，覆盖该地区周围的LN </li><li>清除脂肪组织覆盖和周围的淋巴结，直到微血管床暴露。毗邻的LN的主要动脉段规定，通常由小静脉重叠。整个手术大约需要30分钟。 </li></ol> 4。成像和血管分析<ol><li>一旦血管段的利息是根据解剖的范围公开，安全上编制的活体显微镜。请注意，动物仍然明确手术板，而在整个实验期间活体显微镜。体温是通过加热灯和维护，通过直肠温度计测量，在手术节所述。麻醉平面还监测整个实验过程中，如在2.1节中所述。 </li><li>成立灌流和吸线。出现的PSS灌流水库的重心从饲料，成由5％CO 2平衡氮灌注水套加热水库（传阅和循环水浴加热水套），并为10ml的速度向下滴水线/分钟。应采用吸线，继续拉从筹备灌流PSS的距离。请注意，实验前吸线和水套进行彻底清洗和存储每次实验后。这将确保不育之前的下一个实验。 </li><li>检查superfusing PSS的温度。调整温度循环水的浴缸和/或PSS的灌流速度，实现温度〜37 ° C整个准备工作。 </li><li>允许血管与PSS的平衡至少60分钟，使用视频卡尺量化血管直径。通常，血管直径应确定在多点（例如，在40，50和60分钟），以确保船只已趋于稳定。 </li><li>评估使用一个特定的平滑肌激动剂（如去氧肾上腺素，PE）和内皮细胞的特定受体激动剂（如乙酰胆碱，乙酰胆碱）的船只的健康。取决于激动剂激动剂浓度应调整，但PE和ACH船只应在评估每个浓度累积增加（10 - 5M 10 - 9M）的superfusate。每个受体激动剂的应用程序应使用PSS的船只，直到返回休息直径（一般为30分钟）一洗相分离。 </li><li>可以进行以下评估血管健康，随后的血管活性药物，荧光标记，并跟踪实验细胞。 </li><li>实验结束后，动物过量戊巴比妥钠。动物监测，以确保到脚趾捏和缺乏运动呼吸或心脏跳动，没有响应。在这样的时间，这是颈椎错位。 </li></ol> 5。代表性的成果： 继平衡期的准备工作应产生清晰的图像，可以提供的腹股沟淋巴结的动脉和小静脉识别。应该有最小的脂肪细胞周围的船只，以便清楚地观察到的视频叠加计算血管内径卡尺的小动脉和小静脉的墙壁。一个成功的准备整点是动脉有丰富的血液供应，通过腔和血管张力有一个显着的程度（即动脉有能力vasoconstrict vasodilate平滑肌和内皮细胞的特定受体激动剂分别） 。 <img src="/files/ftp_upload/2551/2551fig1.jpg" alt="图1"/> 图1。腹股沟淋巴结动脉饲料喂养淋巴结平衡与生理盐水和沿端运行的主要微静脉（绿色箭头）（红色箭头）的活体显微镜图像。 <img src="/files/ftp_upload/2551/2551fig2.jpg" alt="图2"/> 图2。累计除了生理盐水灌流苯肾上腺素（PE）的浓度正常的血管活性物质的反应 10-9米至10 -5 M 。一个强大的血管收缩的存在是正常的动脉平滑肌功能目前的暗示。 <img src="/files/ftp_upload/2551/2551fig3.jpg" alt="图3"/> 图3。累计此外，从浓度生理盐水灌流乙酰胆碱（ACH）的正常血管活性反应10-9米至 10 -5 M。一个强大的血管舒张的存在是正常的动脉血管内皮功能目前的暗示。

<ol>
	<li>Bearden, S. E., Payne, G. W., Chisty, A., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arteriolar+network+architecture+and+vasomotor+function+with+ageing+in+mouse+gluteus+maximus+muscle.">Arteriolar network architecture and vasomotor function with ageing in mouse gluteus maximus muscle.</a> J Physiol. 561, 535-545 (2004).</li><li>Looft-Wilson, R. C., Payne, G. W., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexin+expression+and+conducted+vasodilation+along+arteriolar+endothelium+in+mouse+skeletal+muscle.">Connexin expression and conducted vasodilation along arteriolar endothelium in mouse skeletal muscle.</a> J Appl Physiol. 97, 1152-1158 (2004).</li><li>Payne, G. W., Madri, J. A., Sessa, W. C., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histamine+inhibits+conducted+vasodilation+through+endothelium-derived+NO+production+in+arterioles+of+mouse+skeletal+muscle.">Histamine inhibits conducted vasodilation through endothelium-derived NO production in arterioles of mouse skeletal muscle.</a> Faseb J. 18, 280-286 (2004).</li><li>Smeda, J. S., Payne, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+autoregulatory+and+myogenic+function+in+the+cerebrovasculature+of+Dahl+salt-sensitive+rats.">Alterations in autoregulatory and myogenic function in the cerebrovasculature of Dahl salt-sensitive rats.</a> Stroke. 34, 1484-1490 (2003).</li><li>de With, M. C., de Vries, A. M., Kroese, A. B., van der Heijden, E. P., Bleys, R. L., Segal, S. S., Kon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+anatomy+of+the+hamster+retractor+muscle+with+regard+to+its+microvascular+transfer.">Vascular anatomy of the hamster retractor muscle with regard to its microvascular transfer.</a> Eur Surg Res. 42, 97-105 (2009).</li><li>Domeier, T. L., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electromechanical+and+pharmacomechanical+signalling+pathways+for+conducted+vasodilatation+along+endothelium+of+hamster+feed+arteries.">Electromechanical and pharmacomechanical signalling pathways for conducted vasodilatation along endothelium of hamster feed arteries.</a> J Physiol. 579, 175-186 (2007).</li></ol>

这里介绍的节点的腹股沟淋巴结的活体显微镜图像的淋巴结在体内的微血管的能力。因此，它有利于直接，实时观测的一种手段。号法律公告微血管成像是一个独特的的网站，允许一个研究免疫反应和血管之间的接口。使用该制剂，可重点针对在免疫反应，在血管的改变，或在两者之间的互动。 与所有的实验方法，标准活体显微镜的优势和局限。 IVM的标准，如准备在这里描述，可以很容易地进行修改，和作者先前已证明，允许通过引进荧光示踪染料或标记的细胞群啶显微镜。虽然标准IVM的不给三维成像的可能性和跟踪如将双光子显微镜或血管造影，细胞追踪仍然在两个方面取得，使细胞与细胞和细胞对血管的互动要观察和量化，并结合在体内的管理和随后用荧光抗体染色，可用于蛋白/标记表达的实时数据。 值得注意的是，替代的成像技术，需要一个静态图像的环境，经常需要手术区或另外一个在平坦的准备盖玻片夹紧。这限制了，如果不消除，积极灌注血管或其他介质的准备，以评估血管的完整性和生理等，或使用其他技术，如IVM的编制内进行血管扩张实验能力。此外，IVM的标准并不需要一个完全平坦的准备，而不是通过运动，如动物呼吸产生的的影响。这使得更多的外科手术领域，更大的可重复性使用标准的IVM来。这是体现腹股沟淋巴结这里描述的准备。由于淋巴结的大小和形状，不能作准备平整，无组织受伤和节点的位置产生重大的议案，由于动物的呼吸。这些问题将通过其他方法难以克服的，但很容易使用标准IVM的准备的处理。 总之，上面详细的准备，可与任意数量的其他生化，血管和/或转让激活的子集或标记的细胞，缺氧诱导血管介质损耗过度表达，如免疫学技术。然而，更多的应用程序都依赖于前期准备工作的健康。因此，至关重要的是要始终测试成像和评估血管健康。实现健康准备的关键点是确保编制不断灌注平衡PSS的体温和最大限度地减少手术区放置在手术过程中的接触和压力。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th></tr><tr><td>碳酸氢钠</td><td>西格玛爱秩序</td></tr><tr><td>氯化钠</td><td>西格玛爱秩序</td></tr><tr><td>氯化钾</td><td>西格玛爱秩序</td></tr><tr><td>二水氯化钙</td><td>西格玛爱秩序</td></tr><tr><td>硝普钠</td><td>西格玛爱秩序</td></tr><tr><td>去氧</td><td>西格玛爱秩序</td></tr><tr><td>乙酰胆碱</td><td>西格玛爱秩序</td></tr><tr><td>镁氯化亚铁</td><td>西格玛爱秩序</td></tr><tr><td>钠Pentobarbitol </td><td></td></tr></tbody></table>

intravital microscopy of the inguinal lymph node

淋巴结（LN），位于整个身体，免疫系统的一个不可分割的组成部分。他们作为诱导适应性免疫应答的网站，因此，发展效应细胞。因此，LNS打击入侵的病原体，并保持健康的关键。 LN的选择研究是出于可访问性和所需的模型;以及位于腹股沟淋巴结和轻松地支持皮肤和生殖器黏膜感染的生物相关模型的研究。 腹股沟号法律公告，像所有的LNS，拥有广泛的微血管网络，提供血液。在一般情况下，这种微血管网络号法律公告，随后分支机构和饲料高内皮微静脉（HEV）的主要饲料动脉。混合动力汽车是专门用于促进在双方的动态平衡和感染免疫细胞的贩运到的LN。混合动力车如何调节这两种情况下的贩运到的LN是一个激烈的勘探领域。 LN饲料动脉，直接对混合动力汽车的上游影响，主要是营养和丰富的血细胞的LN的供应。此外，在饲料中动脉的变化有牵连在促进适应性免疫反应的诱导。号法律公告微血管，保持一个最佳的血液供应的LN和调节免疫细胞的大量涌入，这是正确的LN函数和随后的免疫反应中的关键要素进入LN的重要性显而易见。 号法律公告在体内的微血管的能力，研究阐明，免疫系统如何和微血管相互作用和影响的LN内的的关键。在这里，我们提出了一个在体内成像的腹股沟淋巴结方法。我们注重的LN微血管成像，要特别注意方法，确保健康血管的研究，​​保持可行的船只数量超过一个小时的成像能力，以及船只幅度量化。还介绍了灌注微血管与血管活性药物，以及潜在的跟踪和量化蜂窝流量的方法。 活体显微镜腹股沟号法律公告允许直接评价微血管功能和实时接口的免疫细胞之间的LN和微循环，的直接接口。这种技术的潜力结合，与许多免疫学技术和荧光细胞标记以及操纵研究其它LNS血管。

Watch this Scientific Journal Video about Intravital Microscopy of the Inguinal Lymph Node at JoVE.com

Intravital Microscopy of the Inguinal Lymph Node

一个执行的腹股沟淋巴结（LN），活体显微镜技术概述。这种技术可以实时，<em&gt;在体内</em&gt;研究淋巴结微血管和结构都在稳态和感染。这种技术可以适应细胞贩运研究和其他淋巴结网站。

腹股沟淋巴结的活体显微镜

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

intravital-microscopy-of-the-inguinal-lymph-node

Research

JoVE Journal

Immunology and Infection

20.0K 次观看.  University of Northern British Columbia. 一个执行的腹股沟淋巴结（LN），活体显微镜技术概述。这种技术可以实时，在体内研究淋巴结微血管和结构都在稳态和感染。这种技术可以适应细胞贩运研究和其他淋巴结网站。

视频: Intravital Microscopy of the Inguinal Lymph Node

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

体外广角T细胞在小鼠淋巴结切片和共聚焦显微镜成像

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

科研

免疫与感染

Murine Superficial Lymph Node Surgery

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often followed over time. Similarly, the study of lymphocyte homeostasis requires time course experiments. Performing these studies within the same mouse is ideal to reduce the experimental variability as well as the number of mice used. Blood withdrawal allows performance of time course experiments, but it only gives information about circulating lymphocytes and provides a limited number of cells1-4. Since lymphocytes circulating through the body and residing in the lymph nodes have different properties, it is important to examine both locations. The sequential removal of lymph nodes by surgery provides a unique opportunity to follow an immune response or immune cell expansion in the same mouse over time. Furthermore, this technique yields between 1-2x106 cells per lymph node which is sufficient to perform phenotypic characterization and/or functional assays. Sequential lymph node surgery or lymphadenectomy has been successfully used by us and others5-11. Here, we describe how the brachial and inguinal lymph nodes can be removed by making a small incision in the skin of an anesthetised mouse. Since the surgery is superficial and done rapidly, the mouse recovers very quickly, heals well and does not experience excessive pain. Every second day, it is possible to harvest one or two lymph nodes allowing for time course experiments. This technique is thus suitable to study the characteristics of lymph node-residing lymphocytes over time. This approach is suitable to various experimental designs and we believe that many laboratories would benefit from performing sequential lymph node surgeries.

To follow the progression of an immune response over time within the same mouse, lymph nodes can be sequentially removed by surgery. Here, we describe how this technique can be performed.

小鼠浅表淋巴结手术

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

活体成像的鼠标使用2 - 光子显微镜的胸腺

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

活体显微镜的脾：寄生虫的流动性和血流量的定量分析

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

小鼠腘窝淋巴结的活体成像

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

淋巴结脂肪垫对嵌合体的淋巴结基质细胞起源研究的一代

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

生发中心的高度已解决活体条纹照明显微镜

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

鼠淋巴结基质细胞的分离

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various biological events in real time and in live animals. This technology has greatly advanced our understanding of physiological processes and pathogen-mediated phenomena in specific organs.
In this study, IVM is applied to the mouse liver and protocols are designed to image in vivo the circulatory system of the liver and measure red blood cell (RBC) velocity in individual hepatic vessels. To visualize the different vessel subtypes that characterize the hepatic organ and perform blood flow speed measurements, C57Bl/6 mice are intravenously injected with a fluorescent plasma reagent that labels the liver-associated vasculature. IVM enables in vivo, real time, measurement of RBC velocity in a specific vessel of interest. Establishing this methodology will make it possible to investigate liver hemodynamics under physiological and pathological conditions. Ultimately, this imaging-based methodology will be important for studying the influence of L. donovani infection&#160;on hepatic hemodynamics.
This method can be applied to other infectious models and mouse organs and might be further extended to pre-clinical testing of a drug&#8217;s effect on inflammation by quantifying its effect on blood flow.

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

肝脏活体如下显微成像<em&gt;利什曼原虫</em&gt;感染：肝脏血流动力学的评估

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and E. coli, and are useful for the study of pathogenesis and/or spread of the bacteria in natural hosts. With the key function of lymph nodes in the host immune response, lymph node tissues serve as a potential source of RNA for downstream transcriptomic analyses, in order to assess the temporal changes in gene expression in cells over the course of an infection. This article presents an overview of the process of lymph node collection, tissue sampling, and downstream RNA processing in livestock, using cattle (Bos taurus) as a model, with additional examples provided from the American bison (Bison bison). The protocol includes information about the location, identification, and removal of lymph nodes from multiple key sites in the body. Additionally, a biopsy sampling methodology is presented that allows for a consistency of sampling across multiple animals. Several considerations for sample preservation are discussed, including the generation of RNA suitable for downstream methodologies like RNA-sequencing and RT-PCR. Due to the long delays inherent in large animal vs. mouse time course studies, representative results from bison and bovine lymph node tissues are presented to describe the time course of the degradation in this tissue type, in the context of a review of previous methodological work on RNA degradation in other tissues. Overall, this protocol will be useful to both veterinary researchers beginning transcriptome projects on large animal samples and to molecular biologists interested in learning techniques for in vivo tissue sampling and in vitro processing.

This protocol provides an overview of procedures for the isolation of RNA for the transcriptomic profiling of lymph node tissues from large animals, including steps in the identification and excision of lymph nodes from livestock and wildlife, sampling approaches to provide consistency across multiple animals, and considerations plus representative results for the post-collection preservation and processing for RNA analysis.

大动物淋巴结的采集与处理 RNA 分析: 大动物 Transcriptomic 淋巴结的制备研究

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and ...