在早期鸡胚跟踪形态组织变形
Summary
本文介绍了表面的标签和<em>前OVO</em>在早期鸡胚组织培养。时间推移明场，荧光，和光学相干断层扫描成像技术适合。高时空分辨率的跟踪表面标签，使运动的数量，如在二维和三维计算的形态株（变形）。
Abstract
胚胎上皮细胞进行复杂的变形（如弯曲，扭曲，折叠和拉伸），形成早期胚胎的原始器官。跟踪基准对这些细胞的表表面标记是一个估算，如生长，收缩和剪切形态数量以及建立的方法。然而，并非所有的表面标记技术容易适应传统的成像方式，具有不同的优势和局限性。在这里，我们描述了两种标记方法，并说明每种技术的效用。在第一种方法，荧光标记的数百个应用同时使用铁磁性颗粒的胚胎。然后，这些标签使用量的2 – D组织变形过程中的形态发生。在第二种方法中，聚苯乙烯微球用于非侵入性光学相干断层扫描（OCT）成像造影剂跟踪的3 – D的组织变形。这些技术已成功LY落实在我们的实验室studythe初头倍，心脏和大脑发育，物理机制，并应适应范围广的形态发生过程。
Protocol
1。一般实验准备准备在层流罩组织培养基。 稀贝科改良Eagle培养基（DMEM）（1大号瓶4.5克/ L葡萄糖，碳酸氢钠，和L -谷氨酰胺）。加入10 mL青霉素/链霉素/新霉素抗生素。 取出用无菌移液管的DMEM 100毫升和100毫升鸡血清取代。 分装DMEM/10％的雏鸡血清/ 1％抗生素无菌的15 mL锥形瓶和冻结。 准备磷酸盐缓冲液（PBS）补充钙和镁。混合100毫升10X PBS中，去离子水9…
Discussion
前OVO文化早期鸡胚两种组织标签技术。首先使用通过铁磁性颗粒的亲脂性荧光染料，同时标签数百个细胞。然而，这种方法是目前不兼容光学相干断层扫描，荧光染料一般使用10月10日从周围组织的对比度很小。因此，我们使用聚苯乙烯微球标记时间推移华侨城分析组织的一种替代技术。这种技术产生的3 – D数据集，但必须小心不逐出组织的珠子。在适当的实验环境中使用这些方法?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作是由国立卫生研究院拨款R01 GM075200和R01 HL083393（LAT）的支持。我们承认从NIH T90的DA022871和Mallinckrodt放射研究所曝气生物滤池的奖学金支持，并从美国心脏协会09PRE2060795授予VDV值。
Materials
| Material / Reagent | Company | Catalogue number | Comments |
| DMEM – high glucose | Sigma-Aldrich | D5796 | |
| Penicillin/Streptomycin/Neomycin | Sigma-Aldrich | P4083 | |
| Chicken Serum | Invitrogen | 16110-082 | |
| Dulbecco’s Phosphate Buffered Saline | Sigma-Aldrich | D1408 | 10X |
| Whatman #2 Filter Paper | Whatman | 1002 090 | 90mm diameter |
| Glass Micropipettes | World Precision Instruments (WPI) | TW150-6 | 1.5mm inner diameter |
| DiI | Invitrogen | D-282 | |
| Iron Reduced | Mallinckrodt Baker Inc. | 5320 | |
| 10 μm Diameter Microspheres (black) | Polysciences Inc. | 24294 | |
| Delta T Dish (for time lapse culture) | Bioptechs | 04200415B | 0.17mm thick, black |
| Delta T4 Culture Dish Controller | Bioptechs | 0420-4-03 | |
| Mini-Pump Variable Flow Device | Fisher Scientific |
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Tags
Early Chick EmbryoEmbryonic EpitheliaBendingTwistingFoldingStretchingPrimitive OrgansTracking Fiducial MarkersMorphogenetic QuantitiesGrowthContractionShearSurface Labeling TechniquesImaging ModalitiesFluorescent LabelsMagnetic Iron ParticlesPolystyrene MicrospheresContrast AgentsNon-invasive Optical Coherence Tomography (OCT) ImagingPhysical MechanismsHead Fold DevelopmentHeart DevelopmentBrain Development
Citar este artigo
Filas, B. A., Varner, V. D., Voronov, D. A., Taber, L. A. Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo. J. Vis. Exp. (56), e3129, doi:10.3791/3129 (2011).