标签胚胎小鼠血管的方法
Summary
本文介绍了标签胚胎皮肤和胸腺血管的方法。
Abstract
功能的血管网络的建立是一个器官形成的重要组成部分，是最佳的器官功能要求。例如，在胸腺适当的血管形成和图案是胸腺细胞进入到器官和成熟的T细胞向周边出口所必需的。在胸腺血管的空间安排，是依赖于从局部微环境的信号，即胸腺上皮细胞（TEC）。最近的一些报告表明，这些信号结果在胸腺血管缺陷1,2中断。以前的研究已经描述技术用于标签的新生儿和成人的胸腺血管 1,2 。我们在这里展示一个标签血管在胚胎胸腺技术。这种方法结合使用FITC -葡聚糖或加纳（Bandeiraea）Simplicifolia凝集素我（1 – GSL isolectin乙4）面部静脉注射和CD31抗体染色，以确定胸腺的血管结构和PDGFR -β标签胸腺血管周围间质 3-5 。还提供了使用冰冻切片或vibratome部分选项。该协议可用于识别胸腺血管的缺陷，这是定义在胸腺血管形成的TEC -派生分子的角色的关键。作为方法的标签，整个血管，它也可以被用来分析多个器官和整个组织，包括皮肤和心脏 6-10胚胎的血管网络。
Protocol
1。荧光标记的葡聚糖和金思朗我isolectin乙面部静脉注射标签胚胎血管准备FITC -葡聚糖（50ug/mL）在磷酸盐缓冲液（PBS）或金思朗1 – isolectin乙4（20ug/200uL）在1.5ml的Eppendorf管中，并在PBS温暖至37 ° C 。股票1.25MM快速绿/ PBS 100uL FITC -葡聚糖溶液（总体积毫升）和股票1.25MM快速180uL绿/ PBS金思朗1 – isolectin乙4（总体积200uL），这样的解决方案是明显的蓝色。 解剖E14.5 – E18.5胚胎…
Discussion
全安装和PECAM – 1（CD31）染色部分标签在胚胎小鼠的血管的常规方法。这些方法需要使用的直接和/或间接免疫荧光和洗涤剂通透小鼠组织。这被证明是一个相当及时处理。在这里，我们有FITC -葡聚糖或isolectin B级4面部静脉注射直接标记的胚胎血管，从而消除了抗体标记的步骤要求。此外，这种方法使血管功能状态的评估，将通过这种方法，而不是由传统抗体染色方法显示“漏水”的表型。…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作是支持授权号码R01AI055001和R01AI082127从NIAID的自然资源管理和SREB论文奖学金奖JLB。
Materials
| Name of the reagent | Company | Catalogue number |
|---|---|---|
| FITC-dextran | Sigma | FD150S-1G |
| Fluorescein labeled GSL 1 – isolectin B4 | Vector Laboratories | FL-1201 |
| Fast Green | MP Biomedicals | 195178 |
| PFA | Fluka | 76240 |
| Fetal Bovine Serum | Atlanta Biologicals | S11550 |
| Optimal Cutting Temperature Compound (O.C.T. | VWR | 25608-930 |
| Acetone | JT Baker | 9006-33 |
| Donkey Serum | Jackson | 017-000-121 |
| rat anti-mouse CD31, | BD Pharmingen | 558736 |
| goat anti-mouse PDGFR-β | R&D Systems | AF1042 |
| donkey anti-rat CD31 Alexa 647 (Invitrogen) | Biolegend | 102516 |
| donkey anti-goat Alexa 594 (Invitrogen) | Invitrogen | A11058 |
| Triton X -100 | Sigma-Aldrich | X-100 |
| Low melt agarose/PBS | Sigma-Aldrich | A9414-25G |
| Methanol | Fisher Scientific | A413-4 |
| Benzyl Alcohol | Acros Scientific | 148390010 |
| Benzyl Benzoate | Acros Scientific | 105860010 |
| Depression slides | Fisher Scientific | S175201 |
| Fluorogel | Electron Microscopy Sciences | 17985-10 |
| Cover Glass (22X22)-1.5 | Thermo Scientific | 152222 |
| Zeiss LSM 510 Meta Confocal Microscope | Zeiss | |
| Micro dissecting forceps | Roboz | RS-5135 |
| Parafilm No. OM992 | Fisher Scientific | 13-374-16 |
| 12 and 24 well microplates | Evergreen Scientific | 222-8044-01F |
| Superfrost/Plus Microscope Slides | Fisher Scientific | 12-550-15 |
| 4mL clear vials | National Scientific | B7800-2 |
Referências
- Cuddihy, A. R. VEGF-mediated cross-talk within the neonatal murine thymus. Blood. 113, 2723-2731 (2009).
- Muller, S. M. Gene targeting of VEGF-A in thymus epithelium disrupts thymus blood vessel architecture. Proc. Natl. Acad. Sci. U. S. A. 102, 10587-10592 (2005).
- Muller, S. M. Neural crest origin of perivascular mesenchyme in the adult thymus. J. Immunol. 180, 5344-5351 (2008).
- Foster, K. Contribution of neural crest-derived cells in the embryonic and adult thymus. J. Immunol. 180, 3183-3189 (2008).
- Liu, C. Coordination between CCR7- and CCR9-mediated chemokine signals in prevascular fetal thymus colonization. Blood. 108, 2531-2539 (2006).
- Lavine, K. J. Fibroblast growth factor signals regulate a wave of Hedgehog activation that is essential for coronary vascular development. Genes Dev. 20, 1651-1666 (2006).
- Lavine, K. J., Kovacs, A., Ornitz, D. M. Hedgehog signaling is critical for maintenance of the adult coronary vasculature in mice. J. Clin Invest. 118, 2404-2414 (2008).
- Mukouyama, Y. S., Gerber, H. P., Ferrara, N., Gu, C., Anderson, D. J. Peripheral nerve-derived VEGF promotes arterial differentiation via neuropilin 1-mediated positive feedback. Development. 132, 941-952 (2005).
- Mukouyama, Y. S., Shin, D., Britsch, S., Taniguchi, M., Anderson, D. J. Sensory nerves determine the pattern of arterial differentiation and blood vessel branching in the skin. Cell. 109, 693-705 (2002).
- Murphy, P. A. Endothelial Notch4 signaling induces hallmarks of brain arteriovenous malformations in mice. Proc. Natl. Acad. Sci. U. S. A. 105, 10901-10906 (2008).
Tags
Labeling VasculatureEmbryonic MiceBlood Vessel NetworkOrganogenesisThymusVasculature FormationThymocyte EntryMature T-cell ExitBlood Vessel DefectsFITC-dextranGriffonia Simplicifolia Lectin IGSL 1 – Isolectin B4CD31 Antibody StainingThymic Perivascular MesenchymeCryosectionsVibratome SectionsThymus Vascular DefectsTEC-derived MoleculesVascular NetworksMultiple Organs
Citar este artigo
Bryson, J. L., Coles, M. C., Manley, N. R. A Method for Labeling Vasculature in Embryonic Mice. J. Vis. Exp. (56), e3267, doi:10.3791/3267 (2011).