全安装胚胎肢体皮肤血管的免疫组织化学分析：一个模型系统，研究在胚胎血管分支形态发生
Summary
我们介绍整个安装的免疫组织化学和激光扫描共聚焦显微镜分析复杂的小鼠胚胎肢体皮肤血管网的形成多个标签。
Abstract
全安装整个血管成像的免疫组织化学分析的关键是了解分支形态发生的细胞机制。我们已经开发出患肢皮肤血管的模型来研究血管发展，其中一个预先存在的原始毛细血管丛是一个层次的分支血管网络重组。全安装多个标签的共聚焦显微镜允许以及完整的血管内皮细胞，周细胞和平滑肌细胞的细胞成分，包括使用特定的荧光标记，强大的成像。这个患肢皮肤血管模型与遗传研究进展，更好地了解血管的发展和图案的分子机制。患肢皮肤血管模型已被用于研究的分化和图案动脉末梢神经如何提供一个空间模板。此视频文章介绍了一个简单而强大的协议染色完整的血管与血管特异性抗体和荧光抗体，这是适用于我们能够遵循的血管发育过程中的吻合血管的胚胎器官。
Protocol
Discussion
血管系统是胚胎发育过程中以及在成人器官维护和生殖功能的器官发育的关键，因为它提供充足的氧气和养分的器官。适当的血管网，以及建立与血管生成的复杂和多步既存的毛细血管网，是具有高度支化结构和层次结构重组的过程。虽然许多作品已被证明在这些过程中涉及的各种分子，它没有明确如何机关或者其组件提供的信号，以促进血管发展的循序渐进的过程，包括内皮分化和血管分支的?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢与小鼠的繁殖和护理援助和实验室管理K.吉尔。也Mukoyama实验室成员的技术帮助表示感谢。经费是由美国卫生Naitonal研究所院内研究计划。
Materials
Antibodies
Pan-endothelial cell marker
| Antibody | Species | Company | Catalogue # | Working condition |
| PECAM-1 | Armenian hamster (M) | Chemicon | MAB1398Z | 1:100 dilution#1 |
| PECAM-1 | Rat (M) | BD | 553369 | 1:300 dilution |
| VEGFR2 | Rat (M) | eBioscience | 14-5821-82 | 1:200 dilution |
| CD34 | Rat (M) | eBioscience | 13-0341 | 1:300 dilution |
| Collagen IV | Rabbit (P) | AbD Serotec | 2150-1470 | 1:300 dilution#2 |
Arterial endothelial cell marker
| Antibody | Species | Company | Catalogue # | Working condition |
| Neuropilin1 | Rabbit (P) | The Alex Kolodkin’s lab#3 | 1:3000 dilution | |
| Unc5H2 | Goat (P) | R&D | AF1006 | 1:200 dilution |
Venous endothelial cell marker
| Antibody | Species | Company | Catalogue # | Working condition |
| EphB4 | Goat (P) | R&D | AF446 | 1:100 dilution |
Lymphatic endothelial cell marker
| Antibody | Species | Company | Catalogue # | Working condition |
| LYVE-1#4 | Rabbit (P) | Abcam | ab14917 | 1:200 dilution |
| LYVE-1#4 | Rat (M) | MBL | D225-3 | 1:200 dilution |
| Prox-1 | Rabbit (P) | Chemicon | AB5475 | 1:1000 dilution |
| Prox-1 | Goat (P) | R&D | AF2727 | 1:50 dilution |
| Neuropilin2 | Rabbit (P) | Cell Signaling | 3366 | 1;100 dilution |
| Podoplanin | Syrian hamster (M) | Hybridoma Bank | 8.1.1 | 1:200 dilution |
Smooth muscle cell/pericyte marker
| Antibody | Species | Company | Catalogue # | Working condition |
| αSMA-Cy3 | Mouse (M)#5 | Sigma | c-6198 | 1:500 dilution#6 |
| NG2 | Rabbit (P) | Chemicon | AB5320 | 1:200 dilution |
| SM22α | Rabbit (P) | Abcam | ab14106 | 1:200 dilution |
Antibodies forGFP reporter
| Antibody | Species | Company | Catalogue # | Working condition |
| GFP | Rabbit (P) | Invitrogen | A11122 | 1:300 dilution |
| GFP | Rat (M) | Nacalai tesque | 04404-84 | 1:1000 dilution |
| GFP | Chick (P) | Chemicon | P42212 | 1:300 dilution |
Antibodies for LacZ reporter
| Antibody | Species | Company | Catalogue # | Working condition |
| β-gal | Rabbit (P) | MP Biomedical | 55976 | 1:5000 dilution |
| β-gal | Goat (P) | AbD Serotec | 4600-1409 | 1:500 dilution |
| β-gal | Chick (P) | Abcam | ab9361 | 1:200 dilution |
Antibodies for peripheral axon
| Antibody | Species | Company | Catalogue # | Working condition |
| 2H3 | Mouse (M)#7 | Hybridoma Bank | 2H3 | 1:200 dilution |
| Tuj1 | Mouse (M)#8 | Covance | MMS-435P | 1:500 dilution |
| Peripherin | Rabbit (P) | Chemicon | AB1530 | 1:1000 dilution |
Antibodies for migrating Schwann cells
| BFABP | Rabbit (P) | The Thomas Müller’s lab#9 | 1:3000 dilution |
(P): polyclonal antibody, (M): monoclonal antibody
#1: Goat anti-Armenian hamster-Cy3 (Jackson ImmunoResearch 127-165-160) antibody should be used as a secondary antibody.
#2: The Collagen IV antibody can be used to detect blood vessels after in situ hybridization.
#3: The Neuropilin1 antibody is kindly provided by the Alex Kolodkin’s lab in the Johns Hopkins University. Sheep anti-human Neuropilin1 antibody is available in R&D (AF3870), although we have not tested it yet.
#4: The LYVE-1 antibodies also detect a subset of macrophages in the embryonic skin.
#5: The anti-αSMA antibody is mouse IgG2a monoclonal antibody.
#6: The Cy3-conjugated αSMA antibody is incubated for 1 hour at room temperature together with secondary antibodies for other primary antibodies.
#7: 2H3 antibody is mouse IgG1 monoclonal antibody against neurofilament.
#8: Tuj1 antibody is mouse IgG2a monoclonal antibody against Neuron-specific class III beta-tubulin．
#9: The BFABP (brain-specific fatty acid binding protein) antibody is kindly provided by the Thomas Müller’s lab in Max-Delbrück-Center for Molecular Medicine.
References
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