Name: inducing ischemia-reperfusion injury in the mouse ear skin for intravital multiphoton imaging of immune responses
Uploaded: 2016-12-22T15:00:00.000+00:00
Duration: 6 min 29 s
Description: Watch this Scientific Journal Video about Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses at JoVE.com

We thank Thomas Graf for providing us with the LysM-eGFP mice.

所有处理活的动物实验均按照所有相关动物的使用和保护准则和条例进行。 1.荧光记者小鼠的选择<ol><li>使用6至12周龄LysM结构-eGFP的11只小鼠（无偏爱或者男性或女性）。 注意:使用各种细胞特异性荧光报道小鼠能够在体内不同的免疫细胞的可视化。在该菌株中，循环嗜中性粒细胞（GFP 喜细胞），循环单核细胞（GFP LO细胞）和真皮的巨噬细胞（GFP LO细胞）可以被可视。与所使用的摄像参数，只能从GFP阳性嗜中性粒细胞的明亮的信号将被检测到。 注:适合这类肌肤的影像学研究的免疫细胞特异性荧光记者小鼠品系的清单可以参考8被发现。 注意:强烈建议小白鼠用于成像，猪人泰德小鼠更容易产生光损伤。这是因为着色耳朵皮肤是多少（指示组织燃烧）激光诱导斑点更敏感。其结果是，中性粒细胞募集和积累可能即使在稳态8,12被观察到。 </li><li>保持在具有12小时光照 - 黑暗周期无特定病原体（SPF）的条件下的小鼠。 </li></ol> 2.鼠标麻醉<ol><li>麻醉小鼠用腹腔注射氯胺酮-赛拉嗪（8微升克-1体重）的，溶解于无菌水中15毫克mL的混合物-1氯胺酮和1mg毫升-1甲苯噻嗪组成。 </li><li>放置在加热垫的鼠标以保持其体温在37℃下在整个制备过程。通过观察一个缺乏脚趾捏反射检查有足够的麻醉。 注意:在第一个小时后，后续季度剂量的麻醉剂将需要被皮下施用的ð将持续大约每0.5小时。晶须或尾部的抽搐还可以指示麻醉穿着关闭，并且需要补足。 </li><li>使用对眼睛的眼科润滑剂，以防止干燥时的麻醉下。 </li></ol> 3.脱毛<ol><li>仔细申请脱毛霜向背侧小鼠耳的使用棉尖涂药器的上三分之二。 </li><li>在彻底，但温和的方式使用湿棉尖涂药除去霜前3分钟 - 等待2。 注意:不要让脱毛膏停留在小鼠耳太久，因为这可能会诱发炎症13,14。 </li></ol> 4.诱导缺血再灌注损伤的<ol><li>采用镀金，N42级的钕磁铁，12毫米直径×2毫米厚，并用约3000高斯的评价诱导缺血小鼠耳部皮肤。 注意:在这种情况下，磁体的微凹面杰诺TES其北极。 </li><li>插槽磁铁成单个的塑料导轨。 注意:塑料导轨用来缓解高强度磁铁的位置和分离。由于其强大的磁力破损但抵抗力低，不要靠近各个磁体放置对方或其他金属。如果他们拉向对方可能会发生破损和分裂。 </li><li>第一（背部）磁体，使得只有边缘与所述第二（腹侧）磁体（ 图3a）接触的位置 注意:这可以防止磁铁从卡扣在一起它们已被正确地定位之前。 </li><li>定位两个磁体，使得所述腹磁铁平躺在耳朵上（ 图3a）。 注意:缺血诱导之前，确保鼠标保持在37℃和足够的麻醉维持（参见步骤2.2）。 </li><li>一旦准备就绪，精心让磁铁走到了一起（ 图3a </STRONG&gt;）。 注意:对于成像目的，夹紧只使红外和非红外区域可被观察到的耳的一半。 </li><li>缺血1.5小时后，通过加捻彼此磁体远离使用塑料导轨，从而允许再灌注发生移除磁体。 注意:必须小心，以防止耳从当磁体被置于压痕。如果肉眼可见的主要血管立即再灌流（ 即血液立即充满血管）的磁体被删除后不完全缺血是显而易见的。虽然再灌注不去除磁铁后立即发生，血管闭塞只是暂时的。因此，当务之急是要准备老鼠耳朵尽可能迅速地成像。 </li></ol> 5.注射血管贴标代理<ol><li>磁铁取出后，立即静脉给药（通过眼眶或尾静脉注射），伊文思蓝（10毫克毫升-1在PBS或盐水; 1μL摹-1体重）或选择的其他血管标记剂。 注意事项:注射前，确保有足够的麻醉仍然是通过执行温和的捏脚趾保持。 </li></ol> 6.对成像平台耳朵放置<ol><li>切2个胶带长度1.5厘米和1.8厘米宽的。 </li><li>使粘合剂双方同时留下1毫米左右的粘合剂沿其宽度粘在一起。 </li><li>切胶带两种，纵向，耳朵平台上的狭缝内适应其位置。 </li><li>插入约一半此遮蔽胶带通过狭缝，使得粘合剂面朝上。 </li><li>加热垫的鼠标的位置，这样，将被成像的耳朵旁边的遮蔽胶带条。 </li><li>通过使用两个PBS浸湿棉尖涂药，轻轻按压胶条的耳朵。 </li><li>使用带为指导，把小鼠耳通过狭缝，同时调整鼠标朝舞台更近。 </li><li>以除去胶带，第一添加PBS一滴以减少带的粘接性。 </li><li>用细的画笔轻轻地分开遮蔽胶带小鼠耳。 </li><li>通过轻轻滚动过耳湿润的棉尖涂药拼合靠近耳部平台的耳朵。 </li><li>放的PBS盖玻片下方的下降（其被保持在使用润滑脂盖玻片夹持位置; 图2），轻轻地将其放置在耳。如有必要，加满PBS多。 注意:盖玻片​​夹持成像期间增加了稳定性。 </li><li>插入直肠温度探头，并根据制造商的说明将电线连接到加热系统。 注:设置体加热垫的温度升高到37℃，并在耳阶段平台至35℃。 </li></ol> 7.多光子显微镜套装和成像参数注意:此协议使用单光束，多光子显微镜可调（680 - 1080 nm）的钛:萨激光（3.3瓦，800纳米，140飞秒脉冲长度; 80 MHz的重复率）用20X的水物镜（NA = 1.0），用于活体成像研究。 <ol><li>打开图像处理软件。 </li><li>根据制造商的说明校准激光。 </li><li>调整激发波长到950纳米。 注:GFP和伊文思蓝可在950 nm的同时兴奋。 </li><li>预览，使用以下设置:500微米2 scanfield，505点¯x505像素的分辨率，和800赫兹的在一个单一的行扫描的扫描频率。点击"预览"。 </li><li>切换衰减器（激光）电源。确保所有必需信号被拾取，而不摄像视场暴露于过量的激光的功率，这可能会引起热损伤。 注:在低功率衰减器启动，如果增加西尼亚l为暗淡。嗜中性粒细胞将不存在从在早期时间间质点 - 再灌注后，巨噬细胞可被用作一个量规来确定所需的最小的功率，因为​​前者比嗜中性粒细胞的调光器。 注意:如果该步骤是在第一时间内完成，调整在非缺血区，在这里完整血管完整性预期的设置，将促进衰减器功率的设置。在随后的实验中，这些设置不需要太多修饰，除非激光功率是不稳定的。 </li><li>调整PMT设置。 注意:检查与制造商的PMT的最大最佳增益电压。设定PMT电压超过推荐阈值将导致更高的信噪比。一般建议的PMT增益电压设置为推荐的门槛，增加了功率衰减器根据需要应信号过于模糊。 </li><li>选择是在接近成像区域缺血的边缘，其特征在于大量的伊文思蓝的泄漏。 </li><li>收集GFP和使用五十零分之五百二十五带通（BP）和655/40 BP滤波器，分别Evans蓝信号。对于二次谐波产生（SHG）信号真皮隔间内的胶原纤维，用一个四十二分之四百七十五BP滤波器。 </li><li>创建一个文件夹来保存图像的格式是可用的图像分析软件兼容。 </li><li>为了获得，使用以下设置:500微米2 scanfield，505点¯x505像素分辨率和扫描频率400赫兹在一个单一的线扫描。 </li><li>一个100微米的Z堆叠为4微米的步长可重复获得随着时间的推移，在1分钟的间隔，以监测中性粒细胞浸润。 注意:尤其对于白细胞（ 例如，中性粒细胞）具有较高的迁徙速度，即长于1分钟，可能会导致细胞跟踪分析期间困难的间隔。在这种情况下，用户既可以降低交流的厚度quisition堆叠或增大扫描频率。 注意:当在缺血区的需要采集栈可以显示为压缩（或者其特征在于较高的SHG强度）的结果小，随后再灌注会导致引起该耳朵膨胀大规模炎症。在Z方向显著漂移预期。这样，一个大的Z堆叠的采集是必要以容纳漂移。 </li><li>整个成像，补足PBS和水经常保持耳湿润和物镜浸泡在水中。 注:一个典型的实验一般持续2 - 4小时。根据不同的实验设计，并配上良好控制麻醉制度，延长成像的持续时间是可能的。 </li></ol> 8.终止实验<ol><li>根据该机构的机构动物护理和使用委员会（IACUC）通过二氧化碳窒息安乐死鼠标PRocedures。 注:由该机构的法规，规章和准则确定的批准方法总是安乐死鼠标。如果需要重复成像，按住鼠标加热垫，直到麻醉消退。一旦鼠标已经恢复足够的意识，是移动，鼠标返回到笼子里。 </li></ol> 9.图像分析注意:从成像实验产生的数据可以通过不同的软件包进行可视化。 <ol><li>打开成像分析软件。 </li><li>在超越模式，导入文件（打开→选择想要的文件夹→所有文件，点击"打开"）。 </li><li>如果默认的颜色不适用编辑伪彩色（编辑→显示调整→在通道选项卡，选择从调色板中所需的颜色）。 </li><li>调整亮度，对比度和背景（编辑→显示调整→切换最大值和最小值）。 </l我&gt; <li>确保文件的尺寸是否正确（编辑→图像属性→在几何选项卡，勾选体素的大小。在这个协议中，X = 0.99，Y = 0.99和Z = 4）。 注意:通过由像素分辨率除以scanfield尺寸派生X和Y。 Z是每个切片之间的厚度。 注意:这些数据集的输出可以表示为最大投影电影。 注意:需要跟踪分析，以了解它们的体内功能完全表征细胞活性和相互作用。使用该软件可当场功能细胞追踪详细说明可在协议参考15中找到。 注:有量化白细胞迁移的方法很多，详情可以在评论文章中引用16被发现。 </li></ol>

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The authors have nothing to disclose.

意义 IR为皮肤褥疮的主要原因之一。褥疮的早期阶段（I和II）描述的人的皮肤的状态（相对于底层皮下组织和肌肉）。然而，免疫病因的了解仍然缺乏。在这里，我们存在于小鼠耳部皮肤一个简单而强大的IR模式，以消除这一差距。我们通过夹紧两个磁铁和随后的小鼠耳研究磁铁去除（再灌注）后，下游的免疫反应模拟缺血。通过使用磁铁来产生一个固定的时间周期上每个耳朵一个恒压，诱导局部缺血，我们能够确保实验之间的再现性和一致性。 这种非侵入性的IR模式的优势还通过利用活体IMAGIN的一套行之有效的模式在于其表现出的能力，实时，体内的免疫反应克耳的皮肤，这相比于皮肤侧翼过程成像期间提供更多的稳定性。目前的一些皮肤侧面模型涉及到可能会改变皮肤4的免疫景观外科手术。此外，成像皮肤侧翼本身提出了一个技术挑战，因为呼吸动作可能5,6成像过程中有助于运动有关的文物。我们目前的模式规避了这些问题。 除了研究免疫细胞于IR的生理反应，这种模式也可以适用于像糖尿病，其中糖尿病可通过增加氧化应激恶化IR损伤的病理生理的设置。理解免疫反应将有助于伤口愈合的理解。 关键步骤一旦鼠标麻醉下，体温调节受损和核心体温急剧下降。为了防止hypot荷米亚，外部加热是维持核心体温在37℃下是必不可少的。耳，位于远侧到主体核心，是由1生理学冷却器 - 2℃，必须保持在35℃。在加热这种一致性也将确保细胞动力学的可重复的读出超时。同时，加热系统必须进行检查，以确保该鼠标还没有结束加热整个麻醉的整个长度。 使用适合所有皮肤成像研究白化小鼠（BALB / c和C57BL / 6-C 2J）的强烈建议，以避免出现色素引起的斑点受伤。如果白化小鼠不可用，在图像采集期间降低激光功率可以减轻对色素的小鼠的问题。但是，进一步的优化可能是必要的，因为组织穿透的减小的深度可能会导致较差的捕获结果。 耳皮肤很细腻;因此，必须小心，以避免任何情况下该可能会引起炎症。的不当技术的实例包括，但不限于以下:1）在离开脱毛霜上的时间超过建议什么过量; 2）在耳朵上施加过大的摩擦通过耳成像平台的狭缝使耳或拆卸从遮蔽胶带耳朵时时;和3）过热耳，无论是由于一有故障的加热系统或可记录的环境温度，而不是鼠标核心温度的放错位置的温度反馈探针。 修改和故障排除大多数关于耳朵制备过程的故障处理的先前已列出8。如果耳是在稍后的时间点再灌注后成像，耳朵可能会膨胀超出由金属夹中创建的0.5毫米的间隙，以容纳耳（ 图1）的厚度。在这种情况下，盖玻片过耳的位置可能会阻碍血液流动，当肉眼可见的血管从视野中消失这是显而易见的。这必须通过创建盖玻片架和黄铜平台之间有较大的差距来避免。这可以通过两种方式通过手动调节所述盖玻片固定器（ 图2）的高度或通过使用较厚的金属夹子来完成，。 该技术的局限性在这个协议中，我们使用伊文思蓝来确定缺血区的位置，因为在间质中的伊文思蓝的泄漏在早期时间点缺血后标志着缺血区的边界。伊文思蓝通常用作血管标记试剂。然而，炎性条件下，血管完整性受到严重损害。这导致伊文思蓝的漏入间质。在这个模型中，我们观察到的再灌注后不久血管和间质之间的对比度的急剧损失。因此，在STudies，其中白细胞-内皮细胞相互作用都参与其中，我们建议您用不同大小的其他血管标记试剂试验（如葡聚糖等 ）。 未来应用除了利用LysM结构-eGFP的小鼠表现出嗜中性粒细胞对皮肤的IR如何响应，该模型也可以用于研究其他白细胞的反应，均在浸润（单核细胞），并在居民插页（巨噬细胞）的白细胞。这种模式也可以被扩展到其他研究的IR损伤后检查血液和淋巴完整性，以及白细胞 - 内皮相互作用。再加上以可视化的胶原蛋白通过MP-IVM SHG信号的能力，这也将是有趣的监测IR后或伤口愈合过程中胶原蛋白的完整性。总之，我们已经建立了红外用于活体成像的非侵入性的，鲁棒的模型，以研究在小鼠耳部皮肤免疫应答。

时，有一个短暂缺氧由于随后组织（再灌注）的随后重新氧合血流量（缺血）的阻塞发生缺血再灌注损伤（IRI）。在皮肤中，缺血 - 再灌注（IR）被认为是起作用的因素，压力性溃疡的病理生理学，其中长期卧床易患长期医院病人受伤的一个。在这些患者中，无论是在皮肤和下面的肌肉经常暴露于施加在骨突出的区域重压力，导致局部损伤的是，如果不进行治疗，可能会变得坏死1。 参与了IRI的损失是双重的。期间缺血，血管的闭塞导致的氧气输送的急剧下降到组织。这导致的ATP和pH值，其中失活参与细胞代谢ATP酶的减少。反过来，细胞钙含量秒杀，并强调或损坏Ç厄尔发生凋亡或坏死2。细胞内的内容，或者损害相关分子模式（湿气）的发布，像HMGB1，有助于炎症反应3。再灌注过程中出现的第二个侮辱。虽然氧和pH水平在再灌注期间恢复，这会导致产生的活性氧（ROS），从而导致细胞内脂质，DNA和蛋白质的氧化。因此，促炎介质被激活，从而衬托涉及免疫细胞向炎症部位2招募次级炎症反应。而生化事件导致对炎症反应的级联已经很好地描述，所述免疫细胞活动的空间和时间调节都不能很好地理解。 在这里，我们描述了如何使用简单的磁性定位鼠耳皮肤一个强大的IR模式。再加上多光子活体成像（MP-IVM），我们建立了一个模型来研究再灌注发生后发生的体内炎症反应。开发和利用这种技术的基本原理是试图实时了解两者间质和浸润细胞对IR的反应。 使用夹紧技术在皮肤侧面的IR的现有模型是高度侵入性的，因为它们需要在皮肤侧面钢板的手术植入，使其低于理想免疫学研究4。类似的非侵入性的夹紧技术已在小鼠皮肤侧翼5,6进行了描述。但是，由于在该方法中，活体成像成分的掺入，我们改为选择耳朵皮肤作为目标的IR部位，因为它绕过由于呼吸运动和成像7,8-期间提供稳定性。此外，跨越间质白细胞亚群是耳朵皮肤和皮肤侧面之间是相同的，虽然数字和比例可能会略有变化9。从而，耳朵皮肤表示理想成象部位。 此外，从这些IRI模型检索大多数数据是有限的，以宏观评价（溃疡分级）和端点炎症指标10微观分析。利用该模型，嗜中性粒细胞中的荧光报道小鼠的皮肤再灌注后的细胞反应的实时可视化被启用。以前发表的活耳成像模型被利用8与另外的修饰（ 图1，图2）。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Mice strains&lt;/strong&gt;</td></tr><tr><td>Lysozyme-GFP C57BL/6</td><td>Thomas Graf, Center for Genomic Regulation</td><td></td><td></td></tr><tr><td>C57BL/6-C2J</td><td>Jackson Laboratories</td><td>000058</td><td>To be crossed with Lysozyme-GFP to generate albino Lysozyme-GFP for skin imaging</td></tr><tr><td colspan="4">&lt;strong&gt;Reagents&lt;/strong&gt;</td></tr><tr><td>PBS</td><td></td><td></td><td></td></tr><tr><td>Viaflex 0.9% (wt/vol) saline</td><td>Baxter Healthcare</td><td>F8B1323</td><td></td></tr><tr><td>Ketamine (100 mg mL&lt;sup&gt;&amp;minus;1&lt;/sup&gt; ketamine hydrochloride</td><td>Parnell</td><td></td><td>Ketamine is a controlled drug and all relevant local regulations should be followed</td></tr><tr><td>Ilium Xylazil-20 (20&amp;nbsp;mg&amp;nbsp;mL&lt;sup&gt;&amp;minus;1&lt;/sup&gt; xylazine hydrochloride)</td><td>Troy Laboratories</td><td></td><td>Xylazil-20 is a controlled drug and all relevant local regulations should be followed.</td></tr><tr><td>Evans blue (10&amp;nbsp;mg&amp;nbsp;mL&lt;sup&gt;&amp;minus;1&lt;/sup&gt; in PBS or saline)</td><td>Sigma-Aldrich</td><td>46160</td><td></td></tr><tr><td>Ultrapurified water</td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>Insulin syringe with needle</td><td>BD</td><td>328838</td><td></td></tr><tr><td>Transfer pipettes</td><td>Biologix Research Company</td><td>30-0135</td><td></td></tr><tr><td>3 M paper masking tape</td><td>3M</td><td>2214</td><td></td></tr><tr><td>Deckglaser microscope cover glass (22 mm &amp;times; 32 mm)</td><td>Paul Marienfeld</td><td>101112</td><td></td></tr><tr><td>Curved splinter forceps</td><td>Aesculap, B. Braun Melsungen</td><td>BD312R</td><td></td></tr><tr><td>Veet hair removal cream</td><td>Reckitt Benckiser</td><td></td><td></td></tr><tr><td>Medical cotton-tipped applicators</td><td>Puritan Medical Products Company</td><td>806-WC</td><td></td></tr><tr><td>C-fold towels</td><td>Kimberly-Clark</td><td>20311</td><td></td></tr><tr><td>Kimwipes delicate task wipes</td><td>Kimtech Science</td><td>34155</td><td></td></tr><tr><td>Gold-plated, N42-grade neodymium magnets, 12 mm in diameter and 2 mm thick&amp;nbsp;</td><td>first4magnets</td><td>F656S</td><td></td></tr><tr><td>Plastic guide, 10 cm by 1.5 cm (polyvinyl chloride material)</td><td></td><td></td><td>fold in half lengthwise, bind with masking tape and slot magnet in</td></tr><tr><td>High vacuum grease</td><td>Dow Corning</td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Microscope&lt;/strong&gt;</td></tr><tr><td>TriM Scope II single-beam two-photon microscope</td><td>LaVision BioTec</td><td></td><td></td></tr><tr><td>Tunable (680&amp;ndash;1,080 nm) Coherent Chameleon Ultra II One Box Ti:sapphire laser (&amp;ge;3.3 W at 800 nm; pulse length of 140 fs, 80 MHz repetition rate)</td><td>Coherent</td><td></td><td></td></tr><tr><td>Water-dipping objectives (20&amp;times;, NA = 1.0)</td><td>Olympus</td><td>XLUMPLFLN20xW</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Miscroscope filter and mirror sets (for imaging GFP, SHG, Evans Blue)&lt;/strong&gt;</td></tr><tr><td>495 long-pass</td><td>Chroma</td><td>T495LPXR</td><td></td></tr><tr><td>560 lomg-pass</td><td>Chroma</td><td>T560LPXR</td><td></td></tr><tr><td>475/42 band-pass</td><td>Semrock</td><td>FF01-475/42-25</td><td></td></tr><tr><td>525/50 band-pass</td><td>Chroma</td><td>ET525/50m</td><td></td></tr><tr><td>655/40 band-pass</td><td>Chroma</td><td>NC028647</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Skin-imaging stage platform (refer to diagram for assembly)&lt;/strong&gt;</td></tr><tr><td>A metal base plate (126 mm &amp;times; 126 mm &amp;times; 1 mm)</td><td></td><td></td><td></td></tr><tr><td>A brass platform for the ear (79 mm &amp;times; 19 mm; 1 mm thickness at side, 0.5 mm thickness in the middle; &lt;strong&gt;Figure 1&lt;/strong&gt;) with slit (1.7 mm &amp;times; 1 mm; 1.5 mm away from long edge)</td><td></td><td></td><td></td></tr><tr><td>Two plastic blocks (10 mm in height)&amp;mdash;for heat insulation</td><td></td><td></td><td></td></tr><tr><td>Curved holder, for positioning the control thermistor on the ear platform</td><td></td><td></td><td></td></tr><tr><td>Interface cable CC-28 with DIN connector and thermistors, one for the temperature control and the other for the temperature monitor</td><td>(Warner Instruments (Harvard Apparatus)</td><td>640106</td><td>connect the interface cable to both resistive heater blocks set at 35 &amp;deg;C</td></tr><tr><td>Resistive heater blocks RH-2</td><td>(Warner Instruments (Harvard Apparatus)</td><td>640274</td><td>Resistive heater blocks can heat the brass ear platform up to over 100 &amp;deg;C within minutes. Ensure that the control thermistor has been properly secured in the holder in order to avoid overheating.</td></tr><tr><td>Temperature controller TC-344B for the ear platform</td><td>(Warner Instruments (Harvard Apparatus)</td><td>640101</td><td></td></tr><tr><td>Temperature controller TR-200 for mouse heating pad</td><td>Fine Science Tools</td><td>21052-00</td><td>Unit is no longer for sale. Ask manufacturer for alternatives</td></tr><tr><td>Power supply for TR-200</td><td>Fine Science Tools</td><td>21051-00</td><td>Unit is no longer for sale. Ask manufacturer for alternatives</td></tr><tr><td>Heating pad</td><td>Fine Science Tools</td><td>21060-00</td><td>Unit is no longer for sale. Ask manufacturer for alternatives.&amp;nbsp;</td></tr><tr><td>Animal rectal probe&amp;nbsp;</td><td>Fine Science Tools</td><td>21060-01</td><td>Unit is no longer for sale. Ask manufacturer for alternatives. After connecting the rectal probe and heating pad to the temperature controller TR-200, set the temperature to 37 &amp;deg;C</td></tr><tr><td colspan="4">&lt;strong&gt;Coverslip holder&lt;/strong&gt;</td></tr><tr><td>2 plastic rods, 1 cm in diameter, 10 cm in length</td><td></td><td></td><td></td></tr><tr><td>1 plastic adaptor with holes drilled to accommodate rods (refer to diagram)</td><td></td><td></td><td></td></tr><tr><td>3 plastic tightening screws for keeping plastic rods in place</td><td></td><td></td><td></td></tr><tr><td>1 metal plate, 6 cm x 2.5 cm, with a 2 cm square cut at 1 end, 2 mm edge away from short edge</td><td></td><td></td><td></td></tr><tr><td>1 pair of nut and bolt for attaching metal plate to plastic rod</td><td></td><td></td><td></td></tr><tr><td>1 acrylic base (4 cm x 5 cm x 1.5 cm) with magnet to hold coverslip holder on skin-imaging stage platform. 1 rod is permanently fixed onto base.</td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Imaging analysis software&lt;/strong&gt;</td></tr><tr><td>Imaris v8.1.2</td><td>Bitplane</td><td></td><td></td></tr></tbody></table>

inducing ischemia-reperfusion injury in the mouse ear skin for intravital multiphoton imaging of immune responses

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent re-oxygenation of the tissues (reperfusion). In the skin, ischemia-reperfusion (IR) is the main contributing factor to the pathophysiology of pressure ulcers. While the cascade of events leading up to the inflammatory response has been well studied, the spatial and temporal responses of the different subsets of immune cells to an IR injury are not well understood. Existing models of IR using the clamping technique on the skin flank are highly invasive and unsuitable for studying immune responses to injury, while similar non-invasive magnet clamping studies in the skin flank are less-than-ideal for intravital imaging studies. In this protocol, we describe a robust model of non-invasive IR developed on mouse ear skin, where we aim to visualize in real-time the cellular response of immune cells after reperfusion via multiphoton intravital imaging (MP-IVM).

该协议使用一个定制的耳部皮肤成像平台， 如图1。这个平台的几个特点专门设计，以方便成像，同时保持生理的设置。放置耳加热黄铜平台上不仅保持在35℃的生理温度耳，但它也隔离由于呼吸不可避免动作耳。在加入黄铜平台上的金属夹的创建一个间隙，以防止在盖玻片固定器从施加在耳朵上的重量，从而保持不间断的血流量。阶段平台也被设计成容纳一个加热垫，将维持在37℃的小鼠在整个成像过程。 盖玻片夹持器（ 图2）被设计为允许盖玻片被附着在金属保持器与真空格雷亚的帮助本身。盖玻片夹持器被连接到一个支架，其允许在与适配器的帮助下垂直和水平轴灵活调整。这种方式，用户可以先精确地调整盖玻片过耳的位置，并随后通过紧固适配器上的螺丝固定该位置。 在这个协议中，我们已经描述了使用磁体来模拟缺血再灌注。 图3b显示之前的耳朵代表的意见和缺血后立即生效。在生理条件下，主要的血管，可以肉眼可视化。磁体的夹持效果暂时茎血流，这可通过瞬时热烫效应（黑色箭头），磁体被去除时被观察到。 在这个特殊的示范，成像聚焦在缺血区（ 图3c的边缘</STRONG&gt;）。缺血（夹紧）和非缺血之间的这种接口（自由夹持）区通常是由大量的伊文思蓝漏入间质（白色实线箭头）标记，而在早期时间点缺血区不含任何伊凡斯蓝信号（白箭头）（ 图3d）。这为活体成像研究的重要里程碑，并有助于保持独立实验之间的一致性。 数据显示，在响应于一个IR损伤，中性粒细胞浸润出口进入从完整的血管间质衬里缺血区的边缘，并朝损伤部位（ 图3d和电影1）迁移。我们预期嗜中性粒细胞迁移到间质的延迟响应相对于其他炎症模型由于在较早的时间点再灌注后缺乏可行灌注血管。为了充分体现这些细胞ular活动，细胞追踪分析可以采用以了解他们的体内功能。速度，平均位移，和趋化指数是一些可从细胞跟踪分析而获得的电位读奏。 <img alt="图1" src="/files/ftp_upload/54956/54956fig1.jpg" /> 图1.活体多光子成像一个特制的耳部皮肤舞台的示意图。 （a）用其尺寸耳成像级的顶视图。 （二）正视图和尺寸。注意，该黄铜板是在侧面厚，以便提供稳定性的平台，和更薄的中间，以接触面积与皮肤最小化，从而确保在耳折叠在狭缝的边缘不间断血流和休息的平台。弯曲持有人引脚向下反馈探头（三）侧视图。加热垫可以是（未示出）固定在用遮蔽胶带的平台。从李等人 ，纳特Protoc，2012 8修改。 <a href="http://ecsource.jove.com/files/ftp_upload/54956/54956fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图1" src="/files/ftp_upload/54956/54956fig2.jpg" /> 图2.盖玻片持有人的设计;侧面和俯视图。盖玻片夹持器保持在一个固定位置的盖玻片并因此减少的z漂移。 <a href="http://ecsource.jove.com/files/ftp_upload/54956/54956fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图1" src="/files/ftp_upload/54956/54956fig3.jpg" /> 图3.红外模型。 （一）示意图显示磁铁的定位。 （B） </strong&gt;照片表示预缺血（左）和局部缺血后耳（右;右耳）磁铁夹持之后。示出相对于其中磁体被放在成像的区域的耳朵的（C）的示意图。 （D）从表示一个LysM结构-eGFP的小鼠嗜中性粒细胞浸润后再灌注时间推移序列的最大强度的z投影快照。在HH表示经过时间:mm格式。比例尺:100微米。 <a href="http://ecsource.jove.com/files/ftp_upload/54956/54956fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="电影1" src="/files/ftp_upload/54956/54956mov1.jpg" /> 电影1中性粒细胞募集的再灌注损伤。最大投影的时间推移序列示出响应于LysM结构-eGFP的白化小鼠耳部皮肤再灌注局部缺血的1.5小时后嗜中性粒细胞（绿）浸润。在较早的时间点，缺乏伊文思蓝信号（红色）表示缺血后临时血管闭塞。胶原（蓝色，二次谐波生成）。 <a href="http://ecsource.jove.com/files/ftp_upload/54956/a_LyGW_IR_mov.mov" target="_blank">请点击此处观看该视频。 （右键点击下载）。</a>

Watch this Scientific Journal Video about Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses at JoVE.com

Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses

这个协议描述了在使用磁铁的夹紧小鼠耳部皮肤缺血再灌注（IR）模型的诱导。使用定制的活体成像模型，我们研究的体内炎症反应再灌注后。这项技术的发展背后的基本原理是延长白细胞如何皮肤缺血再灌注损伤反应的认识。

在鼠耳皮肤的免疫反应的活体多光子成像诱导缺血再灌注损伤

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent ...

inducing-ischemia-reperfusion-injury-mouse-ear-skin-for-intravital

Nanyang Technological University

Research

JoVE Journal

Medicine

11.2K 次观看.  Agency for Science, Technology and Research (A*STAR), Biopolis. 这个协议描述了在使用磁铁的夹紧小鼠耳部皮肤缺血再灌注（IR）模型的诱导。使用定制的活体成像模型，我们研究的体内炎症反应再灌注后。这项技术的发展背后的基本原理是延长白细胞如何皮肤缺血再灌注损伤反应的认识。 

视频: Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

与落射荧光组织基质成分和双光子显微镜的长期活体免疫荧光成像

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

科研

生物工程

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.

The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses.

在背皮褶商会鼠标的缺血性组织损伤:一个皮瓣模型，探讨急性缺血持续

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

医学

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

在实时麻醉小鼠成像中性粒细胞和单核细胞在肠系膜静脉用活体显微镜

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

免疫与感染

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

成像的CD4 T细胞间质迁移发炎真皮

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal &#8212; used as a measure of priming 2) in vivo neutrophil labeling &#8212; achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

使用IL-1β启动子驱动的红色荧光蛋白记者小鼠中性粒细胞引发的活体成像

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

脂多糖治疗小鼠肝脏免疫应答中白细胞的双光子活体成像

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions between cellular and non-cellular blood components during thrombus formation, reliable studies of the physiology and pathophysiology of thrombosis can only be performed in vivo. Therefore, this article presents an ear model in hairless mice and focuses on the in vivo analysis of microcirculation, thrombus formation, and thrombus evolution. By using intravital fluorescence microscopy and the intravenous (iv) application of the respective fluorescent dyes, a repetitive analysis of microcirculation in the auricle can easily be performed, without the need for surgical preparation. Furthermore, this model can be adapted for in vivo studies of different issues, including wound healing, reperfusion injury, or angiogenesis. In summary, the ear of hairless mice is an ideal model for the in vivo study of skin microcirculation in physiological or pathophysiological conditions and for the evaluation of its reaction to different systemic or topical treatments.

The ear model of the hairless SKH1-Hrhr mouse enables intravital fluorescence microscopy of microcirculation and phototoxic thrombus induction without prior surgical preparation in the examined microvascular bed. Therefore, the ear of the hairless mouse is an excellent in vivo model to study the complex interactions during microvascular thrombus formation, thrombus evolution, and thrombolysis.

活体显微镜及血栓诱导在无毛小鼠的耳垂

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions ...

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

多光子活体成像用于监测小鼠后肢模型中动脉生成过程中白细胞募集

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...

Imaging Neutrophil Migration in the Mouse Skin to Investigate Subcellular Membrane Remodeling Under Physiological Conditions

The study of immune cell recruitment and function in tissues has been a very active field over the last two decades. Neutrophils are among the first immune cells to reach the site of inflammation and to participate in the innate immune response during infection or tissue damage. So far, neutrophil migration has been successfully visualized using various in vitro experimental systems based on uniform stimulation, or confined migration under agarose, or micro-fluidic channels. However, these models do not recapitulate the complex microenvironment that neutrophils encounter in vivo. The development of multiphoton microscopy (MPM)-based techniques, such as intravital subcellular microscopy (ISMic), offer a unique tool to visualize and investigate neutrophil dynamics at subcellular resolutions under physiological conditions. In particular, the ear of a live anesthetized mouse provides an experimental advantage to follow neutrophil interstitial migration in real-time due to its ease of accessibility and lack of surgical exposure. ISMic provides the optical resolution, speed, and depth of acquisition necessary to track both cellular and, more importantly, subcellular processes in 3D over time (4D). Moreover, multi-modal imaging of the interstitial microenvironment (i.e., blood vessels, resident cells, extracellular matrix) can be readily accomplished using a combination of transgenic mice expressing select fluorescent markers, exogenous labeling via fluorescent probes, tissue intrinsic fluorescence, and second/third harmonic generated signals. This protocol describes 1) the preparation of neutrophils for adoptive transfer into the mouse ear, 2) different settings for optimal sub-cellular imaging, 3) strategies to minimize motion artifacts while maintaining a physiological response, 4) examples of membrane remodeling observed in neutrophils using ISMic, and 5) a workflow for the quantitative analysis of membrane remodeling in migrating neutrophils in vivo.

Neutrophil migration relies on the rapid and continuous remodeling of the plasma membrane in response to chemoattractant and its interactions with the extracellular microenvironment. Described herein is a procedure based on Intravital Subcellular Microscopy to investigate the dynamic of membrane remodeling in neutrophils injected in the ear of anesthetized mice.

对小鼠皮肤中的中性粒细胞迁移进行成像，以研究生理条件下的亚细胞膜重塑

The study of immune cell recruitment and function in tissues has been a very active field over the last two decades. Neutrophils are among the first ...

生物学

使用倒置显微镜进行活体成像以跟踪皮肤细胞动力学

Streamlined Intravital Imaging Approach for Long-Term Monitoring of Epithelial Tissue Dynamics on an Inverted Confocal Microscope

Understanding normal and aberrant in vivo cell behaviors is necessary to develop clinical interventions to thwart disease initiation and progression. It is therefore critical to optimize imaging approaches that facilitate the observation of cell dynamics in situ, where tissue structure and composition remain unperturbed. The epidermis is the body&#39;s outermost barrier, as well as the source of the most prevalent human cancers, namely cutaneous skin carcinomas. The accessibility of skin tissue presents a unique opportunity to monitor epithelial and dermal cell behaviors in intact animals using noninvasive intravital microscopy. Nevertheless, this sophisticated imaging approach has primarily been achieved using upright multiphoton microscopes, which represent a significant barrier for entry for most investigators. This study presents a custom-designed, 3D-printed microscope stage insert suitable for use with inverted confocal microscopes, streamlining the long-term intravital imaging of ear skin in live transgenic mice. We believe this versatile invention, which may be customized to fit the inverted microscope brand and model of choice and adapted to image additional organ systems, will prove invaluable to the greater scientific research community by significantly enhancing the accessibility of intravital microscopy. This technological advancement is critical for bolstering our understanding of live cell dynamics in normal and disease contexts.

作者聚焦：开发使用倒置共聚焦显微镜进行体内活体成像的工具 

The protocol presents a new tool to simplify intravital imaging using inverted confocal microscopy.

简化活体成像方法，用于在倒置共聚焦显微镜上长期监测上皮组织动力学

Understanding normal and aberrant in vivo cell behaviors is necessary to develop clinical interventions to thwart disease initiation and progression. It ...