Name: a method for labeling vasculature in embryonic mice
Uploaded: 2011-10-07T13:00:00
Description: Watch this Scientific Journal Video about A Method for Labeling Vasculature in Embryonic Mice at JoVE.com

This work was supported by grant numbers R01AI055001 and R01AI082127 from NIAID to NRM and SREB Dissertation Fellowship Award to JLB.

1. Fluorescein labeled dextran and GSL I-isolectin B4 facial vein injections to label embryonic vasculature
<ol>
	<li>Prepare FITC-dextran (50ug/mL) in Phosphate Buffered Saline (PBS) or GSL 1 - isolectin B4 (20ug/200uL) in PBS in a 1.5mL Eppendorf tube and warm to 37&deg;C. Add 100uL of stock 1.25mM Fast Green/PBS to the FITC-dextran solution (total volume 1mL) and 180uL of stock 1.25mM Fast Green/PBS to the GSL 1 - isolectin B4 (total volume 200uL), so that the solution is visibly blu...

<ol>
	<li>Cuddihy, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-mediated+cross-talk+within+the+neonatal+murine+thymus.">VEGF-mediated cross-talk within the neonatal murine thymus.</a> Blood. 113, 2723-2731 (2009).</li><li>Muller, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+targeting+of+VEGF-A+in+thymus+epithelium+disrupts+thymus+blood+vessel+architecture.">Gene targeting of VEGF-A in thymus epithelium disrupts thymus blood vessel architecture.</a> Proc. Natl. Acad. Sci. U. S. A. 102, 10587-10592 (2005).</li><li>Muller, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+origin+of+perivascular+mesenchyme+in+the+adult+thymus.">Neural crest origin of perivascular mesenchyme in the adult thymus.</a> J. Immunol. 180, 5344-5351 (2008).</li><li>Foster, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+neural+crest-derived+cells+in+the+embryonic+and+adult+thymus.">Contribution of neural crest-derived cells in the embryonic and adult thymus.</a> J. Immunol. 180, 3183-3189 (2008).</li><li>Liu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordination+between+CCR7-+and+CCR9-mediated+chemokine+signals+in+prevascular+fetal+thymus+colonization.">Coordination between CCR7- and CCR9-mediated chemokine signals in prevascular fetal thymus colonization.</a> Blood. 108, 2531-2539 (2006).</li><li>Lavine, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor+signals+regulate+a+wave+of+Hedgehog+activation+that+is+essential+for+coronary+vascular+development.">Fibroblast growth factor signals regulate a wave of Hedgehog activation that is essential for coronary vascular development.</a> Genes Dev. 20, 1651-1666 (2006).</li><li>Lavine, K. J., Kovacs, A., Ornitz, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+signaling+is+critical+for+maintenance+of+the+adult+coronary+vasculature+in+mice.">Hedgehog signaling is critical for maintenance of the adult coronary vasculature in mice.</a> J. Clin Invest. 118, 2404-2414 (2008).</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>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>Murphy, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+Notch4+signaling+induces+hallmarks+of+brain+arteriovenous+malformations+in+mice.">Endothelial Notch4 signaling induces hallmarks of brain arteriovenous malformations in mice.</a> Proc. Natl. Acad. Sci. U. S. A. 105, 10901-10906 (2008).</li></ol>

Whole-mount and PECAM-1 (CD31) staining on sections are the conventional methods for labeling the vasculature in embryonic mice. These methods require the use of direct and/or indirect immunofluorescence, and detergents to permeabilize mouse tissue. This proves to be a rather timely process. Here, we have employed FITC-dextran or isolectin B4 facial vein injections to directly label the embryonic vasculature, thereby eliminating the requirement for antibody labeling steps. Furthermore, this method allows the a...

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
		</tr>
		<tr>
			<td>FITC-dextran</td>
			<td>Sigma</td>
			<td>FD150S-1G</td>
		</tr>
		<tr>
			<td>Fluorescein labeled GSL 1 - isolectin B4	</td>
			<td>Vector Laboratories</td>
			<td>FL-1201</td>
		</tr>
		<tr>
			<td>Fast Green</td>
			<td>MP Biomedicals</td>
			<td>195178</td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>Fluka</td>
			<td>76240</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum		</td>
			<td>Atlanta Biologicals</td>
			<td>S11550</td>
		</tr>
		<tr>
			<td>Optimal Cutting Temperature Compound (O.C.T.</td>
			<td>VWR</td>
			<td>25608-930</td>
		</tr>
		<tr>
			<td>Acetone	</td>
			<td>JT Baker</td>
			<td>9006-33</td>
		</tr>
		<tr>
			<td>Donkey Serum</td>
			<td>Jackson	</td>
			<td>017-000-121</td>
		</tr>
		<tr>
			<td>rat anti-mouse CD31,</td>
			<td>BD Pharmingen</td>
			<td>558736</td>
		</tr>
		<tr>
			<td>goat anti-mouse PDGFR-&beta;	</td>
			<td>R&amp;D Systems</td>
			<td>	AF1042</td>
		</tr>
		<tr>
			<td>donkey anti-rat CD31 Alexa 647 (Invitrogen) 		</td>
			<td>Biolegend</td>
			<td>102516</td>
		</tr>
		<tr>
			<td>donkey anti-goat Alexa 594 (Invitrogen)		</td>
			<td>Invitrogen</td>
			<td>A11058</td>
		</tr>
		<tr>
			<td>Triton X -100		</td>
			<td>Sigma-Aldrich</td>
			<td>X-100</td>
		</tr>
		<tr>
			<td>Low melt agarose/PBS	</td>
			<td>Sigma-Aldrich</td>
			<td>	A9414-25G</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A413-4</td>
		</tr>
		<tr>
			<td>Benzyl Alcohol</td>
			<td>Acros Scientific</td>
			<td>148390010</td>
		</tr>
		<tr>
			<td>Benzyl Benzoate</td>
			<td>Acros Scientific</td>
			<td>105860010</td>
		</tr>
		<tr>
			<td>Depression slides</td>
			<td>Fisher Scientific</td>
			<td>S175201</td>
		</tr>
		<tr>
			<td>Fluorogel </td>
			<td>Electron Microscopy Sciences</td>
			<td>17985-10</td>
		</tr>
		<tr>
			<td>Cover Glass (22X22)-1.5</td>
			<td>Thermo Scientific</td>
			<td>152222</td>
		</tr>
		<tr>
			<td>Zeiss LSM 510 Meta Confocal Microscope</td>
			<td>Zeiss</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Micro dissecting forceps 		</td>
			<td>Roboz </td>
			<td>RS-5135</td>
		</tr>
		<tr>
			<td>Parafilm No. OM992</td>
			<td>Fisher Scientific</td>
			<td>13-374-16</td>
		</tr>
		<tr>
			<td>12 and 24 well microplates</td>
			<td>Evergreen Scientific</td>
			<td>222-8044-01F</td>
		</tr>
		<tr>
			<td>Superfrost/Plus
Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
		</tr>
		<tr>
			<td>4mL clear vials		</td>
			<td>National Scientific</td>
			<td>B7800-2</td>
		</tr>
	</tbody>
</table>

a method for labeling vasculature in embryonic mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects 1,2. Previous studies have described techniques used to label the neonatal and adult thymus vasculature 1,2. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1 - isolectin B4) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-&beta; to label thymic perivascular mesenchyme 3-5. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart 6-10.

Watch this Scientific Journal Video about A Method for Labeling Vasculature in Embryonic Mice at JoVE.com

A Method for Labeling Vasculature in Embryonic Mice

This article describes a method for labeling embryonic skin and thymus blood vessels.

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

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