Name: using a laminating technique to perform confocal microscopy of the human sclera
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German Research Foundation (FOR2240 "(Lymph) Angiogenesis and Cellular Immunity in Inflammatory Diseases of the Eye" to CC and LMH; HE 6743/2-1 and HE 7643/3-1 to LMH; CU47/6-1 to CC), German Cancer Aid (to LMH and CC), GEROK program University of Cologne (to SLS and LMH), and EU COST BM1302 "Joining Forces to Corneal Regeneration" (to CC).

使用人体组织，必须审查和机构审查委员会或等效的批准。这里所描述的工作被批准由当地伦理委员会批准了科学检查。这项工作是根据赫尔辛基宣言进行。人类巩膜标本采自世界各地的捐助者的眼中（最大验尸时间24小时），在科隆大学眼库眼科部，德国获得。 1.实验准备<ol><li>制备96％的乙醇，并在不同的管中的磷酸缓冲盐水（PBS）。制备在含有2％牛血清白蛋白（BSA）的PBS的推荐稀释的第一抗体，并保持在冰上的所有解决方案。准备每个样品100微升。 </li><li>清洁所有乐器和用锋利的手术刀。重复相同的手术刀切割后，改变手术刀。 </li><li>获得1-2 COLIBRI钳，直微解剖剪刀，＃10手术刀或眼科手术刀微羽毛，和四＆G针。 </li><li>包裹铝箔周围聚苯乙烯板或使用软木板上贴上组织。一个双目立体显微镜下工作。如果需要一个层流下工作无菌。 </li></ol> 2.准备巩膜<ol><li>轻轻握住灯泡和角巩膜部分进行打孔环锯术，然后旋转环锯。仔细而均匀地旋转环锯在表面上进行同样切割的圆形。使用15.5毫米大小的环钻。与切弯剪剩下的附件。取出角巩膜环锯术。这将导致一个向前打开灯泡。 <ol><li>把巩膜与开放的部分向上棉签。取出视网膜和葡萄膜，使用镊子COLIBRI拉断从巩膜两个层（视网膜和色素膜）。不要离开在内巩膜色素葡萄膜。用弯剪去除乳头视网膜和葡萄膜。删除REM癌宁结膜，眼外肌，和从表面的巩膜榫的胶囊。 </li></ol></li><li>通过使用直型剪刀和镊子COLIBRI中取出所需要的尺寸巩膜样本。取2厘米左右从不同的地点2大小的巩膜样本。由于规模小，轻轻握住巩膜切出所需的尺寸采用了直板型剪刀广场。角巩膜环钻限定巩膜样品的前缘。 <ol><li>避免与镊子为那里抢到的组织是不适合激光共聚焦显微镜分析区域反复抓。制备所需数目取决于实验的类型的样品（ 例如 ，前方与后方，比较图1）和抗体的使用量。 </li></ol></li><li>把样品在1.5ml管中供以后使用。固定在1.5ml 96％乙醇的样品15分钟，然后把它们洗干净三次，每次5分钟在摇床1.5毫升PBS中。如果冷冻组织合作，进行快速冷冻之前这一步。 </li><li>使用新鲜的组织。但是，如果这是不可能的，卡扣冷冻在液氮巩膜并保持在-20℃以备后用。如果冷冻组织，使用前解冻工作。避免反复冻融循环。 </li><li>保持在PBS中含有1.5毫升5％的BSA在室温下2小时各样品。此步骤导致这对层叠有帮助的样品的膨胀，也可以防止非特异性结合。 </li><li>样品已经膨胀后，准备巩膜广场层压。显微镜， 例如 ，一个双目立体显微镜下工作。贴上后巩膜样本以聚苯乙烯膜包裹在铝箔或使用＆G针状软木板上。 </li><li>弯针的边缘，使它们不与在显微镜下的视野干扰。 </li><li>层压全层巩膜SAMPLES。 <ol><li>首先，使用抱钳COLIBRI前巩膜边缘。然后小心地切开使用＃10解剖刀，所述眼科手术刀微羽毛，或刮刀刀片浅表巩膜的薄层。水平握住手术刀和底层层薄薄地切表面层。 </li><li>从解剖巩膜广场薄薄一层巩膜。这种方法是比得上标准外科手术技术来小梁切除术过程中制备的翼片和应导致30-80微米厚的层（比较图2）。 </li></ol></li><li>通过， 例如 ，50微升PBS中使用移液管加入少量的PBS于组织防止巩膜的干燥。 </li><li>把切层为100微升PBS在96孔板和标签两个方向（向extern和实习生）和层（肤浅和深刻的）， 例如具有防水颜色。 </li><li>重复步骤2.7-2.9，直到组织充分地被碾压。&lt;/ LI&gt; </ol> 3.进行免疫组化<ol><li>加入100μl所需初级抗体的进入推荐稀释。这里， 例如使用光盘31（单克隆小鼠抗人）和LYVE1抗体（兔抗人），二者以1:1的稀释度:100在PBS（含有2％BSA）并在4℃下孵育过夜。来改变在96孔板的培养基，保持吸管到孔的壁上并取出流体。 </li><li>第二天，洗涤样品三次，每次5分钟，用200-300微升的PBS上的振动筛。添加100微升相应的次级抗体;这里使用的山羊抗小鼠异硫氰酸荧光素（FITC）和山羊抗兔花青染料3（Cy3标记），并孵育在室温下将样品1-2小时。稀释二级抗体1:300在含有2％山羊血清的PBS中。 </li><li>再次冲洗样本三次，每次在200-300微升PBS中的5分钟的振动筛。 </li><li>进行核染色， 如: </EM&gt;。 4&#39;，6-二脒基-2-苯基吲哚（DAPI）（稀释1:2000，在每100微升孔孵育在室温下10分钟），然后再洗涤2次在200-300微升PBS中在摇动器。 </li><li>转移样品到显微镜载玻片，将它们嵌入在荧光封固剂1-2滴，加盖玻片，由覆盖有透明清漆的边缘密封它，并储存在4℃以备后用或者直接检查用共焦的幻灯片显微镜。 </li><li>使用共聚焦显微镜（或等同物）来分析样品。使用所需的放大倍率， 例如 10-40X的放大倍率。 </li><li>为了验证样品的厚度，测量用的Z堆叠共聚焦显微镜。可能的巩膜部分的量取决​​于巩膜的位置，随着厚度的不同之平展和向后（比较介绍）。 </li></ol>

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层压人类巩膜是这个组织的执行共聚焦显微镜的方法。在这个过程中的一个关键步骤是使用乙醇代替福尔马林用于固定组织。根据我们的经验，使用乙醇代替福尔马林固定时获得更好的结果。钝解剖刀加剧过程，应避免使用。同样地，应避免巩膜的干涸，因为它的复杂性的方法，降低了图像的质量。 对于免疫组织化学染色，可以使用比这里施加的那些其他抗体。在一般情况?...

巩膜是覆盖眼，它是由致密结缔组织的刚性外层。它有助于保护眼内的结构和保持眼压。因此，巩膜是明确的目标至关重要。它不含淋巴管1,2，从而形成它与自由淋巴-内眼3-7之间的外自由淋巴边界。它也提供了眼外肌附着位点，从而与筋共享解剖相似之处。 因为巩膜主要由I型胶原的致密束和具有型胶原III，IV，V，VI，VIII族 8,9和弹性10,11的较小的数字，这个组织是不容易使用用于免疫组织化学。 解剖学，巩膜可以分为三个主要层:（1）浅血管巩膜外层，实测结膜和眼球筋膜囊下方并朝向侧面和第面对轨道眼睛E回; （2）巩膜间质，巩膜的主要部分;及（3）的薄层fusca的，这是直接位于葡萄膜上方的薄的着色层。我们对巩膜解剖知识主要来源于20 世纪上半叶。在那个时候，研究人员研究了血管的解剖结构主要是利用墨汁注射12和血管铸造13-15。后来，它血管造影研究16-19进行了研究。 自那时以来，旧的技术进行了改进，新的问题被开发出来，使我们能够补充先前的解剖知识。例如，它仅仅是约十年，因为我们有过这样可靠淋巴标记淋巴血管内皮特定透明质酸受体1（LYVE1）20或podoplanin 21。共聚焦显微镜提供了新的可能性，研究不同的TI的解剖学特征眼睛的ssues。它允许将用于分化细胞的标记物或用于细胞有关的血管和其它解剖结构的本地化多个污渍。它提供了一个概述当样品是尺寸较大的，使我们能够通过样品扫描中搜索一个特定的细胞类型的时。同的Z-Stack技术，共聚焦显微镜可以用于样品达100-200微米。巩膜的不同之处的肌肉插入后面0.3毫米和在后极11 1毫米的厚度。由于其厚度和不透明度两者，巩膜不适合用传统的方法共聚焦显微镜。 为了解决这个问题，巩膜组织进行层叠，以允许他们的共聚焦显微镜分析。这项技术是在人类巩膜更好地了解双方的生理和病理情况下非常有用。

<table><tbody><tr><td>96% ethanol</td><td>Merck Chemicals, Darmstadt, Germany</td><td>P075.4</td><td></td></tr><tr><td>binocular stereo microscope&amp;nbsp;</td><td>Motic, Hongkong, China</td><td>n.a</td><td></td></tr><tr><td>26 G needles&amp;nbsp;</td><td>Terumo, Leuven, Belgium</td><td>303800</td><td></td></tr><tr><td>15.5 mm trepan</td><td>Geuder, Heidelberg, Germany</td><td>n.a</td><td></td></tr><tr><td>no.10 scalpel&amp;nbsp;</td><td>Feather, pfm medical, Osaka, Japan</td><td>2E+08</td><td></td></tr><tr><td>ophthalmic scalpel micro feather&amp;nbsp;</td><td>Feather, pfm medical, Osaka, Japan</td><td>no. 7657BR</td><td></td></tr><tr><td>CD 31 antibody (monoclonal mouse anti human)</td><td>Dako, USA</td><td>IR610</td><td></td></tr><tr><td>LYVE1 antibody&amp;nbsp; (polyclonal rabbit anti human)</td><td>Zytomed, Germany</td><td>RBK014-05</td><td></td></tr><tr><td>goat anti mouse FITC antibody</td><td>Sigma Aldrich, Steinheim, Germany</td><td>F0257</td><td></td></tr><tr><td>goat anti rabbit Cy3 antibody</td><td>Dianova, Germany</td><td>111-165-003</td><td></td></tr><tr><td>Goat Serum normal</td><td>Dako, Glostrup, Denmark</td><td>X090710-8</td><td></td></tr><tr><td>DAPI</td><td>Carl Roth GmbH, Karlsruhe, Germany</td><td>6335.1</td><td></td></tr><tr><td>microscope slides&amp;nbsp;</td><td>Engelbrecht, Ederm&amp;uuml;nde, Germany</td><td>WC7695002</td><td></td></tr><tr><td>Coverslips 24 mm x 24 mm</td><td>Th. Gayer, Lohmar, Germany</td><td>7695026</td><td></td></tr><tr><td>DAKO fluorescent mounting medium&amp;nbsp;</td><td>DAKO, USA</td><td>S3023</td><td></td></tr><tr><td>LSM Meta 510 confocal microscopy&amp;nbsp;</td><td>Carl Zeiss AG, Jena, Germany</td><td>n.a</td><td></td></tr></tbody></table>

using a laminating technique to perform confocal microscopy of the human sclera

巩膜是致密结缔组织覆盖和保护眼睛。它主要由致密的胶原束（I型，III，IV，V，VI和VII）的。由于它的自身荧光，不透明，和厚度，它没有被发现适合于共焦显微镜。另一种方法的一个在这里提出，其使用石蜡包埋用于免疫组织化学福尔马林固定巩膜，具有技术挑战，预热抗原修复组织时尤其如此。由于巩膜在两个细胞和血管相对较差，使用较大的组织样本进行了探讨，以帮助防止俯瞰细胞，并了解他们有关的船只及其他解剖部位的定位。以允许共焦显微镜下放大的组织样本的分析，进行层压技术来创建从巩膜薄层。继CD31血管和淋巴管内皮hyalu的结果分析罗南受体1（LYVE1）阳性细胞，获得针对该批准科学检查，该方法的优点和局限性进行了讨论。

在这里进行的实验的代表，也有因使用这种特殊层压技术的衍生论证好处。在第一个实验中示出的巩膜外血管丛的三种代表性的照片（ 图3）的不同的网络。这些船只是积极为CD31。 第二个实验中显示了免疫细胞，在巩膜和它们对CD31阳性血管关系的特定LYVE1 +细胞。这里是在10X放大率用的Z-Stack技术穿过组织进行扫描...

Watch this Scientific Journal Video about Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera at JoVE.com

Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera

Human sclera tissue is mainly collagen; therefore, it is not easily usable for immunohistochemistry. To achieve the goal of performing immunohistochemistry for confocal microscopy of scleral tissue, a laminating technique was used.

使用层压技术来执行人类巩膜共聚焦显微镜


The sclera is a dense connective tissue that covers and protects the eye. It mainly consists of dense collagen bundles (types I, III, IV, V, VI, and ...

using-laminating-technique-to-perform-confocal-microscopy-human

Research

JoVE Journal

Medicine

7.5K 次观看.  University of Cologne. Human sclera tissue is mainly collagen; therefore, it is not easily usable for immunohistochemistry. To achieve the goal of performing immunohistochemistry for confocal microscopy of scleral tissue, a laminating technique was used. 

视频: Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Immunolabeled, 澄清人胎盘长柔毛血管网络的三维渲染与分析

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

科研

发育生物学

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

影像引导下，血管源性微流控网络的基于激光的研制

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

生物工程

Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells

In vivo, activation, proliferation, and function of immune cells all occur in a three-dimensional (3D) environment, for instance in lymph nodes or tissues. Up to date, most in vitro systems rely on two-dimensional (2D) surfaces, such as cell-culture plates or coverslips. To optimally mimic physiological conditions in vitro, we utilize a simple 3D collagen matrix. Collagen is one of the major components of extracellular matrix (ECM) and has been widely used to constitute 3D matrices. For 3D imaging, the recently developed light-sheet microscopy technology (also referred to as single plane illumination microscopy) is featured with high acquisition speed, large penetration depth, low bleaching, and photocytotoxicity. Furthermore, light-sheet microscopy is particularly advantageous for long-term measurement. Here we describe an optimized protocol how to set up and handle human immune cells, e.g. primary human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells in the 3D collagen matrix for usage with the light-sheet microscopy for live cell imaging and fixed samples. The procedure for image acquisition and analysis of cell migration are presented. A particular focus is given to highlight critical steps and factors for sample preparation and data analysis. This protocol can be employed for other types of suspension cells in a 3D collagen matrix and is not limited to immune cells.

Here, we present a protocol to visualize immune cells embedded in a three-dimensional (3D) collagen matrix using light-sheet microscopy. This protocol also elaborates how to track cell migration in 3D. This protocol can be employed for other types of suspension cells in the 3D matrix.

三维人体免疫细胞可视化的光片显微术

In vivo, activation, proliferation, and function of immune cells all occur in a three-dimensional (3D) environment, for instance in lymph nodes or ...

免疫与感染

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy include photochemical cross-linking and tissue cross-linking (TXL) methods. Such methods for inducing mechanical tissue property changes are being employed to the cornea in corneal thinning (mechanically weakened) disorders such as keratoconus as well as the sclera in progressive myopia, where thinning and weakening of the posterior sclera occurs and likely contributes to axial elongation. The primary target proteins for such tissue strengthening are fibrillar collagens which constitute the great majority of dry weight proteins in the cornea and sclera. Fortuitously, fibrillar collagens are the main source of second harmonic generation signals in the tissue extracellular space. Therefore, modifications of the collagen proteins, such as those induced through cross-linking therapies, could potentially be detected and quantitated through the use of second harmonic generation microscopy (SHGM). Monitoring SHGM signals through the use of a laser scanning microscopy system coupled with an infrared excitation light source is an exciting modern imaging method that is enjoying widespread usage in the biomedical sciences. Thus, the present study was undertaken in order to evaluate the use of SHGM microscopy as a means to measure induced cross-linking effects in ex vivo rabbit sclera, following an injection of a chemical cross-linking agent into the sub-Tenon&#39;s space (sT), an injection approach that is standard practice for causing ocular anesthesia during ophthalmologic clinical procedures. The chemical cross-linking agent, sodium hydroxymethylglycinate (SMG), is from a class of cosmetic preservatives known as formaldehyde releasing agents (FARs). Scleral changes following reaction with SMG resulted in increases in SHG signals and correlated with shifts in thermal denaturation temperature, a standard method for evaluating induced tissue cross-linking effects.

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking TXL for Myopia

This protocol describes techniques for evaluating chemical cross-linking of the rabbit sclera using&#160;second harmonic generation imaging and differential scanning calorimetry.

兔巩膜二次谐波信号作为治疗组织交叉连接 (txl-) 评价近视的工具

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy ...

医学

<meta charset="utf8"></head><body>用于上皮细胞和成纤维细胞的先进双层 3D 共培养系统

Establishing a Three-Dimensional Coculture Module of Epithelial Cells Using Nanofibrous Membranes

Technical hurdles in a culture of epithelial cells include dedifferentiation and loss of function. Biomimetic three-dimensional (3D) cell culture methods can enhance cell culture efficiency. This study introduces an advanced two-layered culture system intended to cultivate epithelial cells as tissue-like layers with the culture of fibroblasts within a 3D environment. Polyvinyl alcohol (PVA) and poly(&#949;-caprolactone) (PCL) nanofibrous membranes (NMs) were fabricated via electrospinning and utilized as a physiologically relevant extracellular matrix for the culture of epithelial cells and fibroblasts, respectively. In the upper insert wells, lung epithelial cells were cultivated on the PVA NM, and in the lower chambers, fibroblasts were cultured on the PCL NM. This configuration eliminates direct cell-cell contact and facilitates the examination of paracrine signaling mediated by soluble factors. Confocal microscopy was employed to analyze the distribution, growth pattern, and expression of intracellular proteins, including zona occludens in epithelial cells. Z-stacking techniques enabled detailed 3D reconstructions, providing precise insights into the integrity of tight junctions and spatial organization within the epithelial layer. Scanning electron microscopy (SEM) assessed the morphological characteristics of cell types on the nanofibrous membranes. SEM imaging revealed intricate cell surface structures and interactions with the nanofibers, offering a comprehensive perspective on cellular architecture and cell interaction with nanofibrous structure. The Cell Counting Kit-8 (CCK-8) assay is a simple method for measuring epithelial cell and fibroblast growth rates over time. It provides the proliferative behaviors and potential synergistic effects of coculturing these cells. These findings highlight the effectiveness of a simple insert co-culture system for simultaneous culture of fibroblasts and epithelial cells, which is crucial in various physiological and pharmacological contexts, including epithelial tissue regeneration, tumor microenvironment with endothelial, immune, and other stroma cells, toxicity assay, and drug activity test.

<meta charset="utf8"></head><body>作者聚焦:使用 PVA-PCL 纳米纤维膜推进 3D 共培养系统

Here, we demonstrate how epithelial cells cultured with fibroblasts in the nanofibrous membrane-based two-layer system can stably adhere to and grow on the membrane.

使用纳米纤维膜建立上皮细胞的三维共培养模块

Technical hurdles in a culture of epithelial cells include dedifferentiation and loss of function. Biomimetic three-dimensional (3D) cell culture methods ...

Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic nerve and retinal ganglion cell (RGC) degeneration, resulting in visual dysfunction by interrupting the transmission of visual information from the eye to the brain. Elevated intraocular pressure is the most important risk factor; thus, several models of ocular hypertension have been developed in rodents by either genetic or experimental approaches to investigate the causes and effects of the disease. Among those, some limitations have been reported such as surgical invasiveness, inadequate functional assessment, requirement of extensive training, and highly variable extension of retinal damage. The present work characterizes a simple, low-cost, and efficient method to induce ocular hypertension in rodents, based on low-temperature, full-circle cauterization of the limbal vascular plexus, a major component of aqueous humor drainage. The new model provides a technically easy, noninvasive, and reproducible subacute ocular hypertension, associated with progressive RGC and optic nerve degeneration, and a unique post-operative clinical recovery rate that allows in vivo functional studies by both electrophysiological and behavioral methods.

The goal of this protocol is to characterize a novel model of glaucomatous neurodegeneration based on 360&#176; thermic cauterization of limbal vascular plexus, inducing subacute ocular hypertension.

角膜缘血管丛全圆烧灼治疗啮齿动物手术诱发的青光眼

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic ...

神经科学

A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging from neural circuits to subcellular neuronal structures are visualized in mouse brain slices optically cleared with ScaleSF. This clearing method is a modified version of ScaleS and is a hydrophilic tissue clearing method for tissue slices that achieves potent clearing capability as well as a high-level of preservation of fluorescence signals and structural integrity. A customizable three dimensional (3D)-printed imaging chamber is designed for reliable mounting of cleared brain tissues. Mouse brains injected with an adeno-associated virus vector carrying enhanced green fluorescent protein gene were fixed with 4% paraformaldehyde and cut into slices of 1-mm thickness with a vibrating tissue slicer. The brain slices were cleared by following the clearing protocol, which include sequential incubations in three solutions, namely, ScaleS0 solution, phosphate buffer saline (&#8211;), and ScaleS4 solution, for a total of 10.5&#8211;14.5 h. The cleared brain slices were mounted on the imaging chamber and embedded in 1.5% agarose gel dissolved in ScaleS4D25(0) solution. The 3D image acquisition of the slices was carried out using a confocal laser scanning microscope equipped with a multi-immersion objective lens of a long working distance. Beginning with mesoscopic neuronal imaging, we succeeded in visualizing fine subcellular neuronal structures, such as dendritic spines and axonal boutons, in the optically cleared brain slices. This protocol would facilitate understanding of neuronal structures from circuit to subcellular component scales.

The protocol provides a detailed method of neuronal imaging in brain slice using a tissue clearing method, ScaleSF. The protocol includes brain tissue preparation, tissue clarification, handling of cleared slices and confocal laser scanning microscopy imaging of neuronal structures from mesoscopic to microscopic levels.

一种从介观尺度到微观尺度的神经元成像组织清除方法

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging ...

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult mice for in vivo longitudinal real-time monitoring of thymocytes&#180; dynamics within a vascularized thymus segment. Following the transplantation, laser scanning microscopy (LSM) through the cornea allows in vivo noninvasive repeated imaging at cellular resolution level. Importantly, the approach adds to previous intravital T-cell maturation imaging models the possibility for continuous progenitor cell recruitment and mature T-cell egress recordings in the same animal. Additional advantages of the system are the transparency of the grafted area, permitting macroscopic rapid monitoring of the implanted tissue, and the accessibility to the implant allowing for localized in addition to systemic treatments. The main limitation being the volume of the tissue that fits in the reduced space of the eye chamber which demands for lobe trimming. Organ integrity is maximized by dissecting thymus lobes in patterns previously shown to be functional for mature T-cell production. The technique is potentially suited to interrogate a milieu of medically relevant questions related to thymus function that include autoimmunity, immunodeficiency and central tolerance; processes which remain mechanistically poorly defined. The fine dissection of mechanisms guiding thymocyte migration, differentiation and selection should lead to novel therapeutic strategies targeting developing T cells.

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

The goal of the protocol is to show longitudinal intravital real-time tracking of thymocytes by laser scanning microscopy in thymic implants in the anterior chamber of the mouse eye. The transparency of the cornea and vascularization of the graft allows for continuously recording progenitor cell recruitment and mature T-cell egress.

激光扫描显微术实时跟踪眼球前室胸腺细胞

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult ...

使用层压技术来执行人类巩膜共聚焦显微镜

摘要

探索更多视频

此视频中的章节

Immunolabeled, 澄清人胎盘长柔毛血管网络的三维渲染与分析

影像引导下，血管源性微流控网络的基于激光的研制

三维人体免疫细胞可视化的光片显微术

兔巩膜二次谐波信号作为治疗组织交叉连接 (txl-) 评价近视的工具

使用纳米纤维膜建立上皮细胞的三维共培养模块

使用纳米纤维膜建立上皮细胞的三维共培养模块

使用纳米纤维膜建立上皮细胞的三维共培养模块

角膜缘血管丛全圆烧灼治疗啮齿动物手术诱发的青光眼

一种从介观尺度到微观尺度的神经元成像组织清除方法

激光扫描显微术实时跟踪眼球前室胸腺细胞