Summary

标签胚胎小鼠血管的方法

Published: October 07, 2011
doi:

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 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。…

Divulgaciones

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

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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).

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