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.</li>
 <li>Separate and dissect the embryo. Remove the very thin amnion from the embryo.</li>
 <li>(Option) Dissect a single embryo in a 35 x 10 mm Petri dish if each embryo needs to be genotyped. Dissected tail is transferred to a 0.2 ml PCR tube for genotyping.</li>
 <li>Cut off the forelimbs of embryo and transfer forelimbs by a ring forceps into 24 well plate containing 2 ml of ice-cold fresh 4% Paraformaldehyde (PFA) in Phosphate Buffer Saline (PBS).</li>
 <li>Fix the forelimbs with gentle mixing on the Nutator Mixer at 4&deg;C overnight.</li>
 <li>On the following day, remove the PFA and wash three times for 5 min in 2 ml of PBS with gentle mixing on the Nutator Mixer at room temperature.</li>
 <li>Transfer the forelimbs in 100% methanol (MeOH) and store them at -20&deg;C enzyme freezer (the freezer with critical temperature control and without automatic defrost function). Primary antibodies listed in Table 1 work after the 100% MeOH treatment.</li>
 <li>Peel off skin from the forelimb using fine tweezers. Limb skin, when dehydrated, should separate easily from the limb. First place the limb with ventral side (palm side) facing up. Then using fine tweezers, cut the skin as shown in the video. Next dissect around the entire limb, peeling the skin off gently without any damage.</li>
</ol>
2. Whole-mount Immunohistochemical Staining of Limb Skins
<ol>
 <li>Rehydrate the limb skins in 5ml polypropylene round-bottom tube by incubating through the graded series of MeOH/PBT (PBS + 0.2%Triton X-100) (75%, 50%, 25%) for 5 min each. Note that exchanging solutions should be careful to avoid damaging the limb skins.</li>
 <li>Wash twice for 5 min in PBT with gentle mixing on the Nutator Mixer at room temperature.</li>
 <li>Block the limb skins with either 10%Goat serum/PBS+0.2%TX100 blocking buffer for goat secondary antibodies or 10%Donkey serum/PBS+0.2%TX100 blocking buffer for donkey secondary antibodies for 2 hours with gentle mixing on the Nutator Mixer at room temperature.</li>
 <li>Place the limb skins on a 35 x 10 mm Petri dish and transfer by a ring forceps into 2ml-microcentrifuge tube with 800&mu;l of primary antibodies (appropriate dilution as listed in Tables) in the blocking buffer (either 10%Goat serum/PBS+0.2%TX100 or 10%Donkey serum/PBS+0.2%TX100). Incubate the limb skins with gentle mixing on the Nutator Mixer at 4&deg;C overnight. Note that multiple primary antibodies derived from different species (e.g., rat monoclonal antibody+ rabbit polyclonal antibody) can be used simultaneously.</li>
 <li>On the following day, place the limb skins on a 35 x 10 mm Petri dish and transfer by a ring forceps into 5ml polypropylene round-bottom tube with 4ml of the washing buffer (either 2%Goat serum/PBS+0.2%TX100 or 2%Donkey serum/PBS+0.2%TX100).</li>
 <li>Wash five times for 15 min with gentle mixing on the Nutator Mixer at room temperature.</li>
 <li>Place the limb skins on a 35 x 10 mm Petri dish and transfer by a ring forceps into 2ml-microcentrifuge tube with 800&mu;l of secondary antibodies in the blocking buffer (either 10%Goat serum/PBS+0.2%TX100 or 10%Donkey serum/PBS+0.2%TX100). Typically we use secondary antibodies from Jackson ImmunoResearch (Cy3, Cy5, 1:300 dilutions) or Invitrogen (Alexa Fluor 488, Alexa Fluor 568, Alexa Fluor 633, 1:250 dilution). Filter the secondary antibody solution using 0.22&mu;m PVDF membrane syringe filters to remove aggregated particles of the secondary antibodies. Incubate the limb skins in the dark or wrapped with aluminum foil for 1 hour with gentle mixing on the Nutator Mixer at room temperature. Note that different fluorescent conjugated secondary antibodies derived from different species can be used simultaneously.</li>
 <li>Place the limb skins on a 35 x 10 mm Petri dish and transfer by a ring forceps into 5ml polypropylene round-bottom tube with 4ml of the washing buffer (either 2%Goat serum/PBS+0.2%TX100 or 2%Donkey serum/PBS+0.2%TX100). Wash five times for 15 min in the dark or wrapped with aluminum foil with gentle mixing on the Nutator Mixer at room temperature.</li>
 <li>(Option for counterstaining against nucleus) Incubate the limb skins with 4ml of the washing buffer (either 2%Goat serum/PBS+0.2%TX100 or 2%Donkey serum/PBS+0.2%TX100) with To-Pro-3 (Invitrogen T3605, 1:3000 dilution) in the dark or wrapped with aluminum foil for 10 min with gentle mixing on the Nutator Mixer at room temperature. Then, wash three times for 5 min with 4ml of the washing buffer (either 2%Goat serum/PBS+0.2%TX100 or 2%Donkey serum/PBS+0.2%TX100) in the dark or wrapped by aluminum foil with gentle mixing on the Nutator Mixer at room temperature. Note that strong and specific staining for nuclei is observed for To-pro-3 in a specific emission (HeNe 633 nm excitation).</li>
</ol>
3. Mounting the Limb Skins on Slide
<ol>
 <li>Place the limb skins on a 35 x 10 mm Petri dish. Remove dusts, crystals, fibers from the inner layer of the skins using fine tweezers under the stereomicroscope with low illumination to avoid extensive photo bleaching.</li>
 <li>Transfer the limb skins to adhesive microscopic slide by a ring forceps. Place the skins with the inner layer lying upward on the slide (i.e. towards coverslip). Flatten the skins carefully using fine tweezers and remove carry-over washing buffer by Kimwipe.</li>
 <li>Mount in anti-fade mounting media without air bubbles. We use 25"x25" coverslip. Cure on a flat surface in the dark (e.g., the samples mounted using ProLong Gold reagent are placed overnight in the dark at room temperature before viewing). For long-term storage, seal the coverslip to the slide and store at 4&deg;C.</li>
</ol>
4. Confocal Microscopy
<ol>
 <li>Set up appropriate lasers for fluorophores. We use Leica TCS SP5 confocal microscope with three laser sources including Argon 488nm (for Alexa Fluor 488 and GFP), DPSS 561nm (for Alexa Fluor 568 and Cy3)and HeNe 633nm (for Alexa Fluor 633, Cy5 and To-Pro-3).</li>
 <li>Use sequential scan tool to avoid or reduce crosstalk in which all dyes in double or triple-stained samples will be excited at the same time. In the sequential scan mode, images will be recorded in a sequential order.</li>
 <li>More general information about fluorescent dyes and lasers for excitation may be founded in "Confocal Microscopy for Biologists" by Hibbs (2004)</li>
</ol>
5. Representative Results
Whole-mount triple-label confocal immnofluorescence microscopy in mouse forelimb skin at E15.5 with antibodies to the pan-endothelial cell marker PECAM-1 (Figure 1A-C, D blue), the neurofilament marker 2H3 (Figure 1A green), and the smooth muscle cell marker &alpha;SMA (Figure 1A, B red) revealed a characteristic branching pattern of &alpha;SMA+ arteries, aligned with 2H3+ peripheral nerves in the limb skin. In addition to blood vessel branching, this limb skin vasculature model is used to study patterning of lymphatic vessel branching using antibodies to the lymphatic endothelial cell marker LYVE-1 (Figure 1D, E red) and Neuropilin2 (NRP2) (Figure 1D, F green).
<img src="/files/ftp_upload/2620/2620fig1.jpg" alt="Figure 1" /> 
Figure 1. (A-C) Arteries align with peripheral nerves in the embryonic limb skin. Whole-mount triple-label confocal immnofluorescence microscopy with antibodies to the pan-endothelial cell marker PECAM-1 (A-C, blue), the neurofilament marker 2H3 (A, green), and the smooth muscle cell marker &alpha;SMA (A and B, red) is shown. At E15.5, 2H3+ peripheral nerves (open arrows) associate with arteries (arrowheads) which are covered by &alpha;SMA+ smooth muscle cells. (D-F) Lymphatic vasculature in the embryonic limb skin. Triple-label confocal immnofluorescence microscopy with antibodies to the pan-endothelial cell marker PECAM-1 (D, blue), the lymphatic endothelial cell marker LYVE-1 (D and E, red) and Neuropilin2 (NRP2) (D and F, green) is shown. Lymphatic vessels are visualized by both LYVE-1 and NRP2, whereas LYVE-1 is also expressed by a subset of macrophages (open arrowheads). Scale bar: 100&mu;m.

<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>

No conflicts of interest declared.

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 ...

<table><tbody><tr><td colspan="4">&lt;p class=&quot;jove_step&quot;&gt;Antibodies&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Pan-endothelial cell marker&lt;/strong&gt;&lt;/p&gt;&lt;table border=&quot;1&quot;&gt; &lt;tbody&gt; &lt;tr&gt; &lt;td&gt;&lt;strong&gt;Antibody&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Species&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Company&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Catalogue # &lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Working condition&lt;/strong&gt;&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;PECAM-1&lt;/td&gt; &lt;td&gt;Armenian hamster (M)&lt;/td&gt; &lt;td&gt;Chemicon&lt;/td&gt; &lt;td&gt;MAB1398Z&lt;/td&gt; &lt;td&gt;1:100 dilution&lt;sup&gt;#1&lt;/sup&gt;&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;PECAM-1&lt;/td&gt; &lt;td&gt;Rat (M)&lt;/td&gt; &lt;td&gt;BD&lt;/td&gt; &lt;td&gt;553369&lt;/td&gt; &lt;td&gt;1:300 dilution&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;VEGFR2&lt;/td&gt; &lt;td&gt;Rat (M)&lt;/td&gt; &lt;td&gt;eBioscience&lt;/td&gt; &lt;td&gt;14-5821-82&lt;/td&gt; &lt;td&gt;1:200 dilution&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;CD34&lt;/td&gt; &lt;td&gt;Rat (M)&lt;/td&gt; &lt;td&gt;eBioscience&lt;/td&gt; &lt;td&gt;13-0341&lt;/td&gt; &lt;td&gt;1:300 dilution&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Collagen IV&lt;/td&gt; &lt;td&gt;Rabbit (P)&lt;/td&gt; &lt;td&gt;AbD Serotec&lt;/td&gt; &lt;td&gt;2150-1470&lt;/td&gt; &lt;td&gt;1:300 dilution&lt;sup&gt;#2&lt;/sup&gt;&lt;/td&gt; &lt;/tr&gt; &lt;/tbody&gt;&lt;/table&gt;&lt;p&gt;&lt;strong&gt;Arterial endothelial cell marker&lt;/strong&gt;&lt;/p&gt;&lt;table border=&quot;1&quot;&gt; &lt;tbody&gt; &lt;tr&gt; &lt;td&gt;&lt;strong&gt;Antibody&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Species&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Company&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Catalogue # &lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Working condition&lt;/strong&gt;&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Neuropilin1&lt;/td&gt; &lt;td&gt;Rabbit (P)&lt;/td&gt; &lt;td&gt;The Alex Kolodkin&amp;rsquo;s lab&lt;sup&gt;#3&lt;/sup&gt;&lt;/td&gt; &lt;td&gt;&lt;/td&gt; &lt;td&gt;1:3000 dilution&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Unc5H2&lt;/td&gt; &lt;td&gt;Goat (P) &lt;/td&gt; &lt;td&gt;R&amp;amp;D&lt;/td&gt; &lt;td&gt;AF1006&lt;/td&gt; &lt;td&gt;1:200 dilution&lt;/td&gt; &lt;/tr&gt; &lt;/tbody&gt;&lt;/table&gt;&lt;p&gt;&lt;strong&gt;Venous endothelial cell marker&lt;/strong&gt;&lt;/p&gt;&lt;table border=&quot;1&quot;&gt; &lt;tbody&gt; &lt;tr&gt; &lt;td&gt;&lt;strong&gt;Antibody&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Species&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Company&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Catalogue # &lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Working condition&lt;/strong&gt;&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;EphB4&lt;/td&gt; &lt;td&gt;Goat (P)&lt;/td&gt; &lt;td&gt;R&amp;amp;D&lt;/td&gt; &lt;td&gt;AF446&lt;/td&gt; &lt;td&gt;1:100 dilution&lt;/td&gt; &lt;/tr&gt; &lt;/tbody&gt;&lt;/table&gt;&lt;p&gt;&lt;strong&gt;Lymphatic endothelial cell marker&lt;/strong&gt;&lt;/p&gt;&lt;table border=&quot;1&quot;&gt; &lt;tbody&gt; &lt;tr&gt; &lt;td&gt;&lt;strong&gt;Antibody&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Species&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Company&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Catalogue # &lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Working condition&lt;/strong&gt;&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;LYVE-1&lt;sup&gt;#4&lt;/sup&gt;&lt;/td&gt; &lt;td&gt;Rabbit (P) &lt;/td&gt; &lt;td&gt;Abcam&lt;/td&gt; &lt;td&gt;ab14917&lt;/td&gt; &lt;td&gt;1:200 dilution&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;LYVE-1&lt;sup&gt;#4&lt;/sup&gt;&lt;/td&gt; &lt;td&gt;Rat (M) &lt;/td&gt; &lt;td&gt;MBL&lt;/td&gt; &lt;td&gt;D225-3&lt;/td&gt; &lt;td&gt;1:200 dilution&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Prox-1&lt;/td&gt; &lt;td&gt;Rabbit (P)&lt;/td&gt; &lt;td&gt;Chemicon&lt;/td&gt; &lt;td&gt;AB5475&lt;/td&gt; &lt;td&gt;1:1000 dilution&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Prox-1&lt;/td&gt; &lt;td&gt;Goat (P)&lt;/td&gt; &lt;td&gt;R&amp;amp;D&lt;/td&gt; &lt;td&gt;AF2727&lt;/td&gt; &lt;td&gt;1:50 dilution&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Neuropilin2&lt;/td&gt; &lt;td&gt;Rabbit (P)&lt;/td&gt; &lt;td&gt;Cell Signaling&lt;/td&gt; &lt;td&gt;3366&lt;/td&gt; &lt;td&gt;1;100 dilution&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Podoplanin&lt;/td&gt; &lt;td&gt;Syrian hamster (M)&lt;/td&gt; &lt;td&gt;Hybridoma Bank&lt;/td&gt; &lt;td&gt;8.1.1&lt;/td&gt; &lt;td&gt;1:200 dilution&lt;/td&gt; &lt;/tr&gt; &lt;/tbody&gt;&lt;/table&gt;&lt;p&gt;&lt;strong&gt;Smooth muscle cell/pericyte marker&lt;/strong&gt;&lt;/p&gt;&lt;table border=&quot;1&quot;&gt; &lt;tbody&gt; &lt;tr&gt; &lt;td&gt;&lt;strong&gt;Antibody&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Species&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Company&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Catalogue # &lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Working condition&lt;/strong&gt;&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;&amp;alpha;SMA-Cy3&lt;/td&gt; &lt;td&gt;Mouse (M)&lt;sup&gt;#5&lt;/sup&gt;&lt;/td&gt; &lt;td&gt;Sigma&lt;/td&gt; &lt;td&gt;c-6198&lt;/td&gt; &lt;td&gt;1:500 dilution&lt;sup&gt;#6&lt;/sup&gt;&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;NG2&lt;/td&gt; &lt;td&gt;Rabbit (P) &lt;/td&gt; &lt;td&gt;Chemicon&lt;/td&gt; &lt;td&gt;AB5320&lt;/td&gt; &lt;td&gt;1:200 dilution&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;SM22&amp;alpha;&lt;/td&gt; &lt;td&gt;Rabbit (P)&lt;/td&gt; &lt;td&gt;Abcam&lt;/td&gt; &lt;td&gt;ab14106&lt;/td&gt; &lt;td&gt;1:200 dilution&lt;/td&gt; &lt;/tr&gt; &lt;/tbody&gt;&lt;/table&gt;&lt;p&gt;&lt;strong&gt;Antibodies for&lt;em&gt;GFP&lt;/em&gt; reporter&lt;/strong&gt;&lt;/p&gt;&lt;table border=&quot;1&quot;&gt; &lt;tbody&gt; &lt;tr&gt; &lt;td&gt;&lt;strong&gt;Antibody&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Species&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Company&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Catalogue # &lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Working condition&lt;/strong&gt;&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;GFP&lt;/td&gt; &lt;td&gt;Rabbit (P) &lt;/td&gt; &lt;td&gt;Invitrogen&lt;/td&gt; &lt;td&gt;A11122&lt;/td&gt; &lt;td&gt;1:300 dilution&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;GFP&lt;/td&gt; &lt;td&gt;Rat (M) &lt;/td&gt; &lt;td&gt;Nacalai tesque&lt;/td&gt; &lt;td&gt;04404-84&lt;/td&gt; &lt;td&gt;1:1000 dilution&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;GFP&lt;/td&gt; &lt;td&gt;Chick (P)&lt;/td&gt; &lt;td&gt;Chemicon&lt;/td&gt; &lt;td&gt;P42212&lt;/td&gt; &lt;td&gt;1:300 dilution&lt;/td&gt; &lt;/tr&gt; &lt;/tbody&gt;&lt;/table&gt;&lt;p&gt;&lt;strong&gt;Antibodies for &lt;em&gt;LacZ&lt;/em&gt; reporter&lt;/strong&gt;&lt;/p&gt;&lt;table border=&quot;1&quot;&gt; &lt;tbody&gt; &lt;tr&gt; &lt;td&gt;&lt;strong&gt;Antibody&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Species&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Company&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Catalogue # &lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Working condition&lt;/strong&gt;&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;&amp;#946;-gal&lt;/td&gt; &lt;td&gt;Rabbit (P) &lt;/td&gt; &lt;td&gt;MP Biomedical&lt;/td&gt; &lt;td&gt;55976&lt;/td&gt; &lt;td&gt;1:5000 dilution&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;&amp;#946;-gal&lt;/td&gt; &lt;td&gt;Goat (P) &lt;/td&gt; &lt;td&gt;AbD Serotec&lt;/td&gt; &lt;td&gt;4600-1409&lt;/td&gt; &lt;td&gt;1:500 dilution&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;&amp;#946;-gal&lt;/td&gt; &lt;td&gt;Chick (P)&lt;/td&gt; &lt;td&gt;Abcam&lt;/td&gt; &lt;td&gt;ab9361&lt;/td&gt; &lt;td&gt;1:200 dilution&lt;/td&gt; &lt;/tr&gt; &lt;/tbody&gt;&lt;/table&gt;&lt;p&gt;&lt;strong&gt;Antibodies for peripheral axon&lt;/strong&gt;&lt;/p&gt;&lt;table border=&quot;1&quot;&gt; &lt;tbody&gt; &lt;tr&gt; &lt;td&gt;&lt;strong&gt;Antibody&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Species&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Company&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Catalogue # &lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Working condition&lt;/strong&gt;&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;2H3&lt;/td&gt; &lt;td&gt;Mouse (M)&lt;sup&gt;#7&lt;/sup&gt;&lt;/td&gt; &lt;td&gt;Hybridoma Bank&lt;/td&gt; &lt;td&gt;2H3&lt;/td&gt; &lt;td&gt;1:200 dilution&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Tuj1&lt;/td&gt; &lt;td&gt;Mouse (M)&lt;sup&gt;#8&lt;/sup&gt; &lt;/td&gt; &lt;td&gt;Covance&lt;/td&gt; &lt;td&gt;MMS-435P&lt;/td&gt; &lt;td&gt;1:500 dilution&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Peripherin&lt;/td&gt; &lt;td&gt;Rabbit (P)&lt;/td&gt; &lt;td&gt;Chemicon&lt;/td&gt; &lt;td&gt;AB1530&lt;/td&gt; &lt;td&gt;1:1000 dilution&lt;/td&gt; &lt;/tr&gt; &lt;/tbody&gt;&lt;/table&gt;&lt;p&gt;&lt;strong&gt;Antibodies for migrating Schwann cells&lt;/strong&gt;&lt;/p&gt;&lt;table border=&quot;1&quot;&gt; &lt;tbody&gt; &lt;tr&gt; &lt;td&gt;BFABP&lt;/td&gt; &lt;td&gt;Rabbit (P)&lt;/td&gt; &lt;td&gt;The Thomas M&amp;#252;ller&amp;rsquo;s lab&lt;sup&gt;#9&lt;/sup&gt;&lt;/td&gt; &lt;td&gt;&lt;/td&gt; &lt;td&gt;1:3000 dilution&lt;/td&gt; &lt;/tr&gt; &lt;/tbody&gt;&lt;/table&gt;&lt;p class=&quot;jove_content&quot;&gt;(P): polyclonal antibody, (M): monoclonal antibody&lt;/p&gt;&lt;p class=&quot;jove_content&quot;&gt;#1: Goat anti-Armenian hamster-Cy3 (Jackson ImmunoResearch 127-165-160) antibody should be used as a secondary antibody.&lt;/p&gt;&lt;p class=&quot;jove_content&quot;&gt;#2: The Collagen IV antibody can be used to detect blood vessels after &lt;em&gt;in situ&lt;/em&gt; hybridization.&lt;/p&gt;&lt;p class=&quot;jove_content&quot;&gt;#3: The Neuropilin1 antibody is kindly provided by the Alex Kolodkin&amp;rsquo;s lab in the Johns Hopkins University. Sheep anti-human Neuropilin1 antibody is available in R&amp;amp;D (AF3870), although we have not tested it yet.&lt;/p&gt;&lt;p class=&quot;jove_content&quot;&gt;#4: The LYVE-1 antibodies also detect a subset of macrophages in the embryonic skin.&lt;/p&gt;&lt;p class=&quot;jove_content&quot;&gt;#5: The anti-&amp;alpha;SMA antibody is mouse IgG2a monoclonal antibody.&lt;/p&gt;&lt;p class=&quot;jove_content&quot;&gt;#6: The Cy3-conjugated &amp;alpha;SMA antibody is incubated for 1 hour at room temperature together with secondary antibodies for other primary antibodies.&lt;/p&gt;&lt;p class=&quot;jove_content&quot;&gt;#7: 2H3 antibody is mouse IgG1 monoclonal antibody against neurofilament.&lt;/p&gt;&lt;p class=&quot;jove_content&quot;&gt;#8: Tuj1 antibody is mouse IgG2a monoclonal antibody against Neuron-specific class III beta-tubulin.&lt;/p&gt;&lt;p class=&quot;jove_content&quot;&gt;#9: The BFABP (brain-specific fatty acid binding protein) antibody is kindly provided by the Thomas M&amp;#252;ller&amp;rsquo;s lab in Max-Delbr&amp;#252;ck-Center for Molecular Medicine.&lt;/p&gt;</td></tr></tbody></table>

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 ...

whole-mount-immunohistochemical-analysis-for-embryonic-limb-skin

Research

JoVE Journal

Biology

17.5K Views.  National Heart, Lung, and Blood Institute, National Institutes of Health. 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. 

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

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.

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 ...

Immunology and Infection

Analysis of Coronary Vessels in Cleared Embryonic Hearts

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard microscopy techniques, due to the scattering of imaging light by the thick heart tissue, as these vessels are localized deep within the walls of the developing heart. As optical clearing of tissues using organic solvents such as BABB (1 part benzyl alcohol to 2 parts benzyl benzoate) has been shown to greatly improve the optical penetration depth that can be achieved, we combined clearance of whole, PECAM1-immunostained hearts, with laser-scanning confocal microscopy, in order to obtain high-resolution images of vessels throughout the entire heart. BABB clearance of embryonic hearts takes place rapidly and also acts to preserve the fluorescent signal for several weeks; in addition, samples can be imaged multiple times without loss of signal. This straightforward method is also applicable to imaging other types of blood vessels in whole embryos.

We present a protocol for the analysis of coronary vessels in whole embryonic murine hearts up to E15.5, using standard immunological staining methods followed by optical clearance and confocal microscopy. This technique enables visualization of blood vessels throughout the entire heart without the need for time-consuming analysis of serial sections.

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard ...

Developmental Biology

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Bioengineering

Kinetic Analysis of Vasculogenesis Quantifies Dynamics of Vasculogenesis and Angiogenesis In Vitro

Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of vasculogenesis has become a central readout of endothelial progenitor cell functionality, and therefore, several attempts have been made to improve both in vitro and in vivo vasculogenesis models. However, standard methods are limited in scope, with static measurements failing to capture many aspects of this highly dynamic process. Therefore, the goal of developing this novel protocol was to assess the kinetics of in vitro vasculogenesis in order to quantitate rates of network formation and stabilization, as well as provide insight into potential mechanisms underlying vascular dysfunction. Application of this protocol is demonstrated using fetal endothelial colony forming cells (ECFCs) exposed to maternal diabetes mellitus. Fetal ECFCs were derived from umbilical cord blood following birth, cultured, and plated in slides containing basement membrane matrix, where they underwent vasculogenesis. Images of the entire slide wells were acquired using time-lapse phase contrast microscopy over 15 hours. Images were analyzed for derivation of quantitative data using an analysis software called Kinetic Analysis of Vasculogenesis (KAV). KAV uses image segmentation followed by skeletonization to analyze network components from stacks of multi-time point phase contrast images to derive ten parameters (9 measured, 1 calculated) of network structure including: closed networks, network areas, nodes, branches, total branch length, average branch length, triple-branched nodes, quad-branched nodes, network structures, and the branch to node ratio. Application of this protocol identified altered rates of vasculogenesis in ECFCs obtained from pregnancies complicated by diabetes mellitus. However, this technique has broad implications beyond the scope reported here. Implementation of this approach will enhance mechanistic assessment and improve functional readouts of vasculogenesis and other biologically important branching processes in numerous cell types or disease states.

Kinetic Analysis of Vasculogenesis Quantifies Dynamics of Vasculogenesis and Angiogenesis In Vitro

Here, we present a protocol for time-lapse imaging and analysis of vasculogenesis in vitro using phase contrast microscopy coupled with the open source software, Kinetic Analysis of Vasculogenesis. This protocol can be applied to quantitatively assess the vasculogenic potential of numerous cell types or experimental conditions that model vascular disease.

Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of ...

Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution

Here, we present a protocol of a whole-mount adult ear skin imaging technique to study comprehensive three-dimensional neuro-vascular branching morphogenesis and patterning, as well as immune cell distribution at a cellular level. The analysis of peripheral nerve and blood vessel anatomical structures in adult tissues provides some insights into the understanding of functional neuro-vascular wiring and neuro-vascular degeneration in pathological conditions such as wound healing. As a highly informative model system, we have focused our studies on adult ear skin, which is readily accessible for dissection. Our simple and reproducible protocol provides an accurate depiction of the cellular components in the entire skin, such as peripheral nerves (sensory axons, sympathetic axons, and Schwann cells), blood vessels (endothelial cells and vascular smooth muscle cells), and inflammatory cells. We believe this protocol will pave the way to investigate morphological abnormalities in peripheral nerves and blood vessels as well as the inflammation in the adult ear skin under different pathological conditions.

Here, we describe a high resolution whole-mount imaging method in the entire adult mouse ear skin, which enables us to visualize branching morphogenesis and patterning of peripheral nerves and blood vessels, as well as immune cell distribution.

Here, we present a protocol of a whole-mount adult ear skin imaging technique to study comprehensive three-dimensional neuro-vascular branching ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Neuroscience

A Patient-Derived Xenograft Model for Venous Malformation

Venous malformation (VM) is a vascular anomaly that arises from impaired development of the venous network resulting in dilated and often dysfunctional veins. The purpose of this article is to carefully describe the establishment of a murine xenograft model that mimics human VM and is able to reflect patient heterogeneity. Hyper-activating non-inherited (somatic) TEK (TIE2) and PIK3CA mutations in endothelial cells (EC) have been identified as the main drivers of pathological vessel enlargement in VM. The following protocol describes the isolation, purification and expansion of patient-derived EC expressing mutant TIE2 and/or PIK3CA. These EC are injected subcutaneously into the back of immunodeficient athymic mice to generate ectatic vascular channels. Lesions generated with TIE2 or PIK3CA-mutant EC are visibly vascularized within 7&#8210;9 days of injection and recapitulate histopathological features of VM patient tissue. This VM xenograft model provides a reliable platform to investigate the cellular and molecular mechanisms driving VM formation and expansion. In addition, this model will be instrumental for translational studies testing the efficacy of novel drug candidates in preventing the abnormal vessel enlargement seen in human VM.

We present a detailed protocol to generate a murine xenograft model of venous malformation. This model is based on the subcutaneous injection of patient-derived endothelial cells containing hyper-activating TIE2 and/or PIK3CA gene mutations. Xenograft lesions closely recapitulate the histopathological features of VM patient tissue.

Venous malformation (VM) is a vascular anomaly that arises from impaired development of the venous network resulting in dilated and often dysfunctional ...

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed in mice is high. Phenotypic characterization of these mouse models also requires the examination of cardiac function and morphology. Echocardiography and magnetic resonance imaging (MRI) are commonly used approaches to characterize cardiac function and morphology in mice. Echocardiographic and MRI equipment specialized for use in small rodents is&#160;expensive and requires a dedicated space. This protocol describes cardiac measurements in mice using a clinical echocardiographic system with a 15 MHz human vascular probe. Measurements are performed on anesthetized adult mice. At least three image sequences are recorded and analyzed for each animal in M-mode in the parasternal short-axis view. Afterwards, cardiac histological examination is performed, and cardiomyocyte diameters are determined on hematoxylin-eosin- or wheat germ agglutinin (WGA)-stained paraffin sections. Vessel density is determined morphometrically after Pecam-1 immunostaining. The protocol has been applied successfully to pharmacological studies and different genetic animal models under baseline conditions, as well as after experimental myocardial infarction by the permanent ligation of the left anterior descending coronary artery (LAD). In our experience, echocardiographic investigation is limited to anesthetized animals and is feasible in adult mice weighing at least 25 g.

Echocardiographic examination is frequently used in mice. Expensive high-resolution ultrasound devices have been developed for this purpose. This protocol describes an affordable echocardiographic procedure combined with histological morphometric analyses to determine cardiac morphology.

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed ...

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

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A Method for Labeling Vasculature in Embryonic Mice

Analysis of Coronary Vessels in Cleared Embryonic Hearts

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Kinetic Analysis of Vasculogenesis Quantifies Dynamics of Vasculogenesis and Angiogenesis In Vitro

Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

A Patient-Derived Xenograft Model for Venous Malformation

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse