Name: whole-mount immunohistochemical analysis for embryonic limb skin vasculature: a model system to study vascular branching morphogenesis in embryo
Uploaded: 2011-05-20T13:00:00
Description: Watch this Scientific Journal Video about Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo at JoVE.com

We thank K. Gill for assistance with mouse breeding and care and for laboratory management. Thanks also to Mukoyama lab members for technical help. Funding was provided by Intramural Research Program of Naitonal Institutes of Health.

1. Collecting Mouse Embryonic Limb Skin (E13.5~E17.5)
<ol>
 <li>Euthanize plugged females by approved procedure. According to our approved animal protocol, the females are euthanized by CO2 exposure and then assured by cervical dislocation. Lay the animal on its absorbent paper towel and soak it thoroughly in 70% EtOH/H2O from a squeeze bottle.</li>
 <li>Dissect the uterus intact and place it in a 100 x 15 mm Petri dish containing ice-cold Hanks' Balanced Salt Solution (HBSS) to wash out blood.</...

<ol>
	<li>Mukouyama, Y. S., Shin, D., Britsch, S., Taniguchi, M., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+nerves+determine+the+pattern+of+arterial+differentiation+and+blood+vessel+branching+in+the+skin.">Sensory nerves determine the pattern of arterial differentiation and blood vessel branching in the skin.</a> Cell. 109, 693-705 (2002).</li><li>Mukouyama, Y. S., Gerber, H. P., Ferrara, N., Gu, C., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+nerve-derived+VEGF+promotes+arterial+differentiation+via+neuropilin+1-mediated+positive+feedback.">Peripheral nerve-derived VEGF promotes arterial differentiation via neuropilin 1-mediated positive feedback.</a> Development. 132, 941-952 (2005).</li><li>Wang, H. U., Chen, Z. F., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+distinction+and+angiogenic+interaction+between+embryonic+arteries+and+veins+revealed+by+ephrin-B2+and+its+receptor+Eph-B4.">Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4.</a> Cell. 93, 741-753 (1998).</li><li>Gerety, S. S., Wang, H. U., Chen, Z. F., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symmetrical+mutant+phenotypes+of+the+receptor+EphB4+and+its+specific+transmembrane+ligand+ephrin-B2+in+cardiovascular+development.">Symmetrical mutant phenotypes of the receptor EphB4 and its specific transmembrane ligand ephrin-B2 in cardiovascular development.</a> Mol Cell. 4, 403-414 (1999).</li></ol>

The vascular system is crucial for organ development during embryogenesis as well as for organ maintenance and reproductive functions in the adults, because it supplies sufficient oxygen and nutrients to the organs. Proper vascular network is well-established with complex and multi-step processes by angiogenesis in which pre-existing capillary network is reorganized with highly branched and hierarchical structures. Although numerous works have been shown that a variety of molecules is involved in these processes, it has ...

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			<div>
			Antibodies
Pan-endothelial cell marker
<table border="1">
 <tbody>
 <tr>
 <td>Antibody</td>
 <td>Species</td>
 <td>Company</td>
 <td>Catalogue # </td>
 <td>Working condition</td>
 </tr>
 <tr>
 <td>PECAM-1</td>
 <td>Armenian hamster (M)</td>
 <td>Chemicon</td>
 <td>MAB1398Z</td>
 <td>1:100 dilution#1</td>
 </tr>
 <tr>
 <td>PECAM-1</td>
 <td>Rat (M)</td>
 <td>BD</td>
 <td>553369</td>
 <td>1:300 dilution</td>
 </tr>
 <tr>
 <td>VEGFR2</td>
 <td>Rat (M)</td>
 <td>eBioscience</td>
 <td>14-5821-82</td>
 <td>1:200 dilution</td>
 </tr>
 <tr>
 <td>CD34</td>
 <td>Rat (M)</td>
 <td>eBioscience</td>
 <td>13-0341</td>
 <td>1:300 dilution</td>
 </tr>
 <tr>
 <td>Collagen IV</td>
 <td>Rabbit (P)</td>
 <td>AbD Serotec</td>
 <td>2150-1470</td>
 <td>1:300 dilution#2</td>
 </tr>
 </tbody>
</table>
Arterial endothelial cell marker
<table border="1">
 <tbody>
 <tr>
 <td>Antibody</td>
 <td>Species</td>
 <td>Company</td>
 <td>Catalogue # </td>
 <td>Working condition</td>
 </tr>
 <tr>
 <td>Neuropilin1</td>
 <td>Rabbit (P)</td>
 <td>The Alex Kolodkin's lab#3</td>
 <td></td>
 <td>1:3000 dilution</td>
 </tr>
 <tr>
 <td>Unc5H2</td>
 <td>Goat (P) </td>
 <td>R&amp;D</td>
 <td>AF1006</td>
 <td>1:200 dilution</td>
 </tr>
 </tbody>
</table>
Venous endothelial cell marker
<table border="1">
 <tbody>
 <tr>
 <td>Antibody</td>
 <td>Species</td>
 <td>Company</td>
 <td>Catalogue # </td>
 <td>Working condition</td>
 </tr>
 <tr>
 <td>EphB4</td>
 <td>Goat (P)</td>
 <td>R&amp;D</td>
 <td>AF446</td>
 <td>1:100 dilution</td>
 </tr>
 </tbody>
</table>
Lymphatic endothelial cell marker
<table border="1">
 <tbody>
 <tr>
 <td>Antibody</td>
 <td>Species</td>
 <td>Company</td>
 <td>Catalogue # </td>
 <td>Working condition</td>
 </tr>
 <tr>
 <td>LYVE-1#4</td>
 <td>Rabbit (P) &nbsp;</td>
 <td>Abcam</td>
 <td>ab14917</td>
 <td>1:200 dilution</td>
 </tr>
 <tr>
 <td>LYVE-1#4</td>
 <td>Rat (M) &nbsp;</td>
 <td>MBL</td>
 <td>D225-3</td>
 <td>1:200 dilution</td>
 </tr>
 <tr>
 <td>Prox-1</td>
 <td>Rabbit (P)</td>
 <td>Chemicon</td>
 <td>AB5475</td>
 <td>1:1000 dilution</td>
 </tr>
 <tr>
 <td>Prox-1</td>
 <td>Goat (P)</td>
 <td>R&amp;D</td>
 <td>AF2727</td>
 <td>1:50 dilution</td>
 </tr>
 <tr>
 <td>Neuropilin2</td>
 <td>Rabbit (P)</td>
 <td>Cell Signaling</td>
 <td>3366</td>
 <td>1;100 dilution</td>
 </tr>
 <tr>
 <td>Podoplanin</td>
 <td>Syrian hamster (M)</td>
 <td>Hybridoma Bank</td>
 <td>8.1.1</td>
 <td>1:200 dilution</td>
 </tr>
 </tbody>
</table>
Smooth muscle cell/pericyte marker
<table border="1">
 <tbody>
 <tr>
 <td>Antibody</td>
 <td>Species</td>
 <td>Company</td>
 <td>Catalogue # </td>
 <td>Working condition</td>
 </tr>
 <tr>
 <td>&alpha;SMA-Cy3</td>
 <td>Mouse (M)#5</td>
 <td>Sigma</td>
 <td>c-6198</td>
 <td>1:500 dilution#6</td>
 </tr>
 <tr>
 <td>NG2</td>
 <td>Rabbit (P) &nbsp;</td>
 <td>Chemicon</td>
 <td>AB5320</td>
 <td>1:200 dilution</td>
 </tr>
 <tr>
 <td>SM22&alpha;</td>
 <td>Rabbit (P)</td>
 <td>Abcam</td>
 <td>ab14106</td>
 <td>1:200 dilution</td>
 </tr>
 </tbody>
</table>
Antibodies forGFP reporter
<table border="1">
 <tbody>
 <tr>
 <td>Antibody</td>
 <td>Species</td>
 <td>Company</td>
 <td>Catalogue # </td>
 <td>Working condition</td>
 </tr>
 <tr>
 <td>GFP</td>
 <td>Rabbit (P) &nbsp;</td>
 <td>Invitrogen</td>
 <td>A11122</td>
 <td>1:300 dilution</td>
 </tr>
 <tr>
 <td>GFP</td>
 <td>Rat (M) &nbsp;</td>
 <td>Nacalai tesque</td>
 <td>04404-84</td>
 <td>1:1000 dilution</td>
 </tr>
 <tr>
 <td>GFP</td>
 <td>Chick (P)</td>
 <td>Chemicon</td>
 <td>P42212</td>
 <td>1:300 dilution</td>
 </tr>
 </tbody>
</table>
Antibodies for LacZ reporter
<table border="1">
 <tbody>
 <tr>
 <td>Antibody</td>
 <td>Species</td>
 <td>Company</td>
 <td>Catalogue # </td>
 <td>Working condition</td>
 </tr>
 <tr>
 <td>&beta;-gal</td>
 <td>Rabbit (P) &nbsp;</td>
 <td>MP Biomedical</td>
 <td>55976</td>
 <td>1:5000 dilution</td>
 </tr>
 <tr>
 <td>&beta;-gal</td>
 <td>Goat (P) </td>
 <td>AbD Serotec</td>
 <td>4600-1409</td>
 <td>1:500 dilution</td>
 </tr>
 <tr>
 <td>&beta;-gal</td>
 <td>Chick (P)</td>
 <td>Abcam</td>
 <td>ab9361</td>
 <td>1:200 dilution</td>
 </tr>
 </tbody>
</table>
Antibodies for peripheral axon
<table border="1">
 <tbody>
 <tr>
 <td>Antibody</td>
 <td>Species</td>
 <td>Company</td>
 <td>Catalogue # </td>
 <td>Working condition</td>
 </tr>
 <tr>
 <td>2H3</td>
 <td>Mouse (M)#7</td>
 <td>Hybridoma Bank</td>
 <td>2H3</td>
 <td>1:200 dilution</td>
 </tr>
 <tr>
 <td>Tuj1</td>
 <td>Mouse (M)#8 &nbsp;</td>
 <td>Covance</td>
 <td>MMS-435P</td>
 <td>1:500 dilution</td>
 </tr>
 <tr>
 <td>Peripherin</td>
 <td>Rabbit (P)</td>
 <td>Chemicon</td>
 <td>AB1530</td>
 <td>1:1000 dilution</td>
 </tr>
 </tbody>
</table>
Antibodies for migrating Schwann cells
<table border="1">
 <tbody>
 <tr>
 <td>BFABP</td>
 <td>Rabbit (P)</td>
 <td>The Thomas M&uuml;ller's lab#9</td>
 <td></td>
 <td>1:3000 dilution</td>
 </tr>
 </tbody>
</table>
(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&amp;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-&alpha;SMA antibody is mouse IgG2a monoclonal antibody.
#6: The Cy3-conjugated &alpha;SMA 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&#65294;
#9: The BFABP (brain-specific fatty acid binding protein) antibody is kindly provided by the Thomas M&uuml;ller's lab in Max-Delbr&uuml;ck-Center for Molecular Medicine.		</div>

whole-mount immunohistochemical analysis for embryonic limb skin vasculature: a model system to study vascular branching morphogenesis in embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

Watch this Scientific Journal Video about Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo at JoVE.com

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

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