可视化线虫角质层结构的亲脂性的重要染料，直接投资收益
Summary
我们提出了一个可视化生活角质层的方法<em> C。线虫</em>使用红色荧光亲脂性染料DII（1,1' – 双十八烷基- 3，3,3'，3' – tetramethylindocarbocyanine高氯酸盐），这是常用的<em> C。线虫</em>可视化环境接触的神经元。这种优化的协议，alae和环形表皮结构是由DII染色和使用复合显微镜观察。
Abstract
角质层的C.线虫是一种高度耐药的结构，周围的动物 1-4外部。角质层不仅可以保护环境的动物，但也决定体形和蠕动4-6次的作用。由表皮细胞分泌的几层组成的角质层，包括最外层的脂质层7。
在角质层的圆周脊称为环空中的模式动物的长度， 目前在8发展的所有阶段。 Alae有纵脊目前是在特定的发展阶段，包括L1的dauer，和成人阶段2,9。影响表皮胶原组织的基因突变可以改变表皮结构和动物的身体形态 5,6,10,11 。虽然使用DIC的光学显微镜复合表皮成像是可行的，目前的方法，突出表皮结构包括荧光12％的转基因表达，抗体染色，电子显微镜1。也被用来标记麦胚凝集素（WGA）的可视化表皮糖蛋白，但受限于解决更精细的表皮结构 14 。使用荧光染料染色表皮表面已被观察到，但从来没有在细节 15的特点。我们提出了一个方法来可视化角质层在现场 C. 线虫使用红色荧光亲脂性染料DII（1,1' -双十八烷基- 3，3,3'，3' – tetramethylindocarbocyanine高氯酸盐），也就是通常用于在 C 线虫可视化环境接触的神经元。这是一个简单可靠的方法，高分辨率的荧光可视化的环空中，alae，外阴，男性的尾巴， 在 C和雌雄同体的尾穗DII染色的优化协议线虫 。
Protocol
1。 DII制备染色准备的20毫克/毫升DII（Biotium公司，公司，海沃德，CA）在DMF原液。 DII是光敏感，所以保护光箔包装DII。 每个人口0.6μLDII股票加入到399.4微升M9的创建工作的DII稀释。这应该给最后的30微克/毫升DII在M9的工作液。这可以扩展多个种群同时染色。盾DII在铝箔包装管（S）。 2。线虫的制备包含一个未被污染的线虫人口使用60毫米的钢板。洗?…
Discussion
这里介绍DII的染色方法，允许一个相对快速和方便的方法来可视化的角质层线虫。再利用和优化常用的图像环境暴露15,17感觉神经元的方法，直接投资收益可用于荧光染色alae和环状结构（图1和2），以及外阴，男性的尾巴，和雌雄同体的尾穗（图3）。我们发现，洗液和时间的影响的直接投资收益的能力，始终染色的角质层（表1，图4）。 DII染色环境暴露的神经元的方法使用两个小?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们要感谢S.塔内加- Bageshwar，K. Beifuss，S. Kedroske，和HC。萧为有益的讨论。这项工作是由从分子和细胞医学TAMHSC系的启动资金。该化合物的范围和旋转盘购部门和TAMHSC院长办公室提供的资金。一些菌株提供了线虫遗传学中心，这是由国家研究资源中心资助。 pRF4（ROL – 6（su1006））答：消防的礼物。
Materials
| Reagents | Synonyms | Company | Catalogue number | Comments |
| DiI | 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate | Biotium, Inc. | 60010 | Stock dilution: 20 mg/mL in DMF working dilution: 30 mg/mL |
| DMF | Dimethylformamide | Sigma-Aldrich, Inc. | D4551 | |
| Triton X-100 |
Octylphenoxypolyethoxyethanol | VWR International, LLC. | EM-9410 | |
| M9 | 22mM KH2PO4, 42mM Na2HPO4, 86mM NaCl, 1mM MgSO4 | |||
| NGM | Nematode growth medium | IPM Scientific, Inc. | 11006-501 | Can be prepared following NGM agar protocol18 |
| Agar-agar | EMD Chemicals, Inc. | 1.01614 | 4% in water | |
| Levamisole | Levamisole hydrochloride | Sigma-Aldrich, Inc. | 31742 | 100 μM – 1 mM levamisole as required |
| Microscope slides | VWR International, LLC. | 16005-106 | ||
| Microscope cover glasses | VWR International, LLC. | 16004-302 | ||
| Compound scope | Carl Zeiss, Inc. | A1m | Use objectives to match the needs of the experiment | |
| TRITC or other compatible filter | Chroma Technology Corp. | 49005 | ET – DSRed (TRITC/Cy3) sputtered filter set |
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Tags
VisualizationCaenorhabditis ElegansCuticular StructuresLipophilic Vital Dye DiICuticleResistant StructureEnvironment ProtectionBody ShapeMotilityEpidermal CellsLipid LayerCircumferential RidgesAnnuliAlaeDevelopment StagesMutationsCollagen OrganizationCompound MicroscopyDIC OpticsFluorescent Transgene ExpressionAntibody StainingElectron MicroscopyWheat Germ Agglutinin (WGA)Cuticular GlycoproteinsFluorescent DyeLive Imaging
Citar este artigo
Schultz, R. D., Gumienny, T. L. Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI. J. Vis. Exp. (59), e3362, doi:10.3791/3362 (2012).