使用层压技术来执行人类巩膜共聚焦显微镜
Summary
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.
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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).
Materials
| 96% ethanol | Merck Chemicals, Darmstadt, Germany | P075.4 | |
| binocular stereo microscope | Motic, Hongkong, China | n.a | |
| 26G needles | Terumo, Leuven, Belgium | 303800 | |
| 15.5mm trepan | Geuder, Heidelberg, Germany | n.a | |
| no.10 scalpel | Feather, pfm medical, Osaka, Japan | 2E+08 | |
| ophthalmic scalpel micro feather | Feather, pfm medical, Osaka, Japan | no. 7657BR | |
| CD 31 antibody (monoclonal mouse anti human) | Dako, USA | IR610 | |
| LYVE1 antibody (polyclonal rabbit anti human) | Zytomed, Germany | RBK014-05 | |
| goat anti mouse FITC antibody | Sigma Aldrich, Steinheim, Germany | F0257 | |
| goat anti rabbit Cy3 antibody | Dianova, Germany | 111-165-003 | |
| Goat Serum normal | Dako, Glostrup, Denmark | X090710-8 | |
| DAPI | Carl Roth GmbH, Karlsruhe, Germany | 6335.1 | |
| microscope slides | Engelbrecht, Edermünde, Germany | WC7695002 | |
| Coverslips 24x24mm | Th. Gayer, Lohmar, Germany | 7695026 | |
| DAKO fluorescent mounting medium | DAKO, USA | S3023 | |
| LSM Meta 510 confocal microscopy | Carl Zeiss AG, Jena, Germany | n.a | |
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