Name: Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo (Video) | JoVE Journal
Uploaded: 2011-05-20T13:00:00.000+00:00
Duration: 9 min 53 s
Description: Learn whole-mount immunohistochemistry for vascular branching morphogenesis in mouse embryonic limb skin. This method enables high-resolution confocal imaging of blood and lymphatic vessel development.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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°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°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 800μl 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°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 800μl 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 0.22μm 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°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 αSMA (Figure 1A, B red) revealed a characteristic branching pattern of αSMA+ 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="Patterning of nerves, blood, lymphatic vessels; immunofluorescence microscopy; NRP2, PECAM-1." data-altupdatedat="1788235205000" data-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 αSMA (A and B, red) is shown. At E15.5, 2H3+ peripheral nerves (open arrows) associate with arteries (arrowheads) which are covered by αSMA+ 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: 100μm.

<ol><li>Mukouyama, Y. S., Shin, D., Britsch, S., Taniguchi, M., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sensory+nerves+determine+the+pattern+of+arterial+differentiation+and+blood+vessel+branching+in+the+skin%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Peripheral+nerve-derived+VEGF+promotes+arterial+differentiation+via+neuropilin+1-mediated+positive+feedback%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">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/pubmed?term=((Molecular+distinction+and+angiogenic+interaction+between+embryonic+arteries+and+veins+revealed+by+ephrin-B2+and+its+receptor+Eph-B4%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Symmetrical+mutant+phenotypes+of+the+receptor+EphB4+and+its+specific+transmembrane+ligand+ephrin-B2+in+cardiovascular+development%5BTitle%5D)+AND+%22Mol+Cell%22%5BJournal%5D)">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 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.

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 ...

The overall goal of this procedure is to perform whole Mount immunohistochemical analysis for imaging the entire vasculature in developing limb skin. This is accomplished by first cutting four limbs from the embryo, fixing the four limbs in 4%paraform aldehyde in PBS, and then dehydrating them in 100%methanol. The second step is to use fine tweezers to peel off the skin at the base of the limb.Follow the blue dashed lines and peel off the skin in a dish containing 100%methanol. Remove the skin from the limb. Next immunohistochemical staining with vascular specific primary antibodies and fluorescent secondary antibodies is performed on the limb skin.Finally, whole mount confocal microscopy with multiple labeling will enable robust imaging of the entire limb skin vasculature That may advantage of this technique over existing methods such as cross count histology analysis. Using embryo sectioning is that whole mount immunohistochemical analysis of limb skin vasculature allows us not only to image the entire vasculature, including the cell components, but also to study processes of differentiation and the patterning of blood vessel cells and the lymphatic vessel branching. Are we demonstrating the procedure together with winding DA postdoc from my laboratory, Embryonic limb skins are collected from pregnant female mice.After removing and washing the uterus, work under a dissecting microscope to separate and dissect the embryos. Once the embryos have been dissected, the next step is to remove the four LIMS from the embryos using fine tweezers. Cut the four lim at the shoulder, then use a ring forceps to transfer the four lim to a 24 well plate containing ice.Cold, fresh 4%Paraform Hyde in PBS place two four LIMS per well in two milliliters of 4%PFA. Fix the four limbs with gentle mixing on a mutator mixer at four degrees Celsius overnight on the following day. Remove the PFA from each well and add two milliliters of PBS.Wash the specimens for five minutes with gentle mixing on the mutator mixer at room temperature. Repeat this wash twice for a total of three washes. Next, transfer the four limbs to a five milliliter polypropylene round bottom tube containing 100%methanol store at minus 20 degrees Celsius in an enzyme freezer with critical temperature control and without an automatic defrost function After overnight storage and the minus 20, the skin can be peeled off from the falling.This is one of the most critical aspects of this procedure because the success of the subsequent staining and imaging depends on not damaging the skins. The limb skin separates easily from the limb when dehydrated in this manner to peel off the skin from the four limb. First place the limb with the ventral side facing up.Then using fine tweezers, dissect through the skin near the base of the limb toward the paw in a straight line. Next, dissect around the entire limb, peeling the skin off as you turn the limb. Once the skins have been peeled off.The four limbs begin whole mount immunohistochemical staining of the limb skins Prepare beforehand. Three stock 50 milliliter tubes with 75%50%and 25%methanol in PBT for rehydrating. The skins, the limb skins are rehydrated by incubating through a grated series of methanol from the highest to the lowest methanol concentrations for five minutes each.To begin transfer the limb skin to a five milliliter polypropylene round bottom tube containing 75%methanol in PBT. After five minutes on the mutator mixer, carefully aspirate the 75%methanol and refill with 50%methanol and PBT. Five minutes later.Replace the 50%methanol with 25%methanol and PBT. At the end of the final incubation. Carefully aspirate the 25%methanol PBT and refill with PBT.Wash the limb skins for five minutes with gentle mixing on the mutator mixer at room temperature. Repeat this wash once. Being careful to avoid damaging the limbs skins during the exchange of solutions.Once the limbs skins have been rehydrated, block them with the appropriate blocking buffer. Incubate for two hours with gentle mixing on the nutt mixer at room temperature After two hours, place the limb skins on a 35 by 10 millimeter Petri dish. Then use ring forceps to transfer the skins into a two milliliter fuge tube containing 800 microliters of primary antibodies diluted in the appropriate blocking buffer.Here, multiple primary antibodies derived from different species can be used simultaneously. Incubate the limbs skins with gentle mixing on the mutator mixer at four degrees Celsius overnight on the following day, place the limbs skins on a 35 by 10 millimeter Petri dish. Transfer the skins into a five milliliter polypropylene round bottom tube containing four liters of the appropriate washing buffer.Wash five times for 15 minutes each with gentle mixing on the mutator mixer at room temperature. Carefully replace the used buffer with new buffer for each wash during the washes. Prepare the secondary antibodies to remove aggregated particles of the secondary antibodies.Use a 0.22 micron PVDF membrane syringe filter to filter the secondary antibody solution into a 1.5 milliliter fuge tube. Then centrifuge the filtered sample at 15, 000 RPM for 10 minutes at four degrees Celsius to remove particles and air bubbles. Collect the filtered antibody solution in a two milliliter micro fuge tube.When the five washes are done, place the limbs skins on a 35 by 10 millimeter Petri dish and transfer them into a two liter micro fuge tube containing 800 microliters of secondary antibodies in the blocking buffer. Different fluorescent conjugated secondary antibodies derived from different species can be used simultaneously in this step. Incubate the limbs, skins in the dark for one hour with gentle mixing on the mutator mixer at room temperature.After the one hour incubation, place the limbs skins on a 35 by 10 millimeter Petri dish and transfer them into a five milliliter polypropylene round bottom tube containing four milliliters of the appropriate washing buffer. Wash five times for 15 minutes each in the dark with gentle mixing on the Tator mixer at room temperature for imaging by confocal microscopy. The limb skins must be mounted on a slide after immunohistochemical staining.To begin this procedure, place the stained limb skins on a 35 by 10 millimeter Petri dish, working under a stereo microscope with low illumination. To avoid extensive photo bleaching, use fine tweezers to remove dust crystals and fibers from the inner layer of the skins. Transfer the limb skins to an adhesive microscopy slide.Place the skins with the inner layer lying upward on the slide facing the cover slip. Then flatten the skins carefully using fine tweezers. Remove carry over washing buffer by Kim wipe.Add Antifa mounting media to the skins and place a 25 by 25 cover slip on top of the specimens. Taking care not to introduce air bubbles cure on a flat surface in the dark at room temperature for long-term storage of the specimens. Seal the cover slip to the slide with nail polish and store at four degrees Celsius.The patterning of nerves and blood vessels in mouse for limb skin at E 15.5 is shown by these representative results of whole mount triple label confocal immunofluorescence microscopy immunofluorescence of the pan endothelial cell marker pcam one is blue. The neurofilament marker two H three is green, and the smooth muscle cell marker alpha SMA is red. These images revealed a characteristic branching pattern of alpha SMA positive arteries indicated by white arrowhead aligned with two H three peripheral nerves indicated by open arrows in addition to blood vessel branching.This limb skin vasculature model can also reveal the patterning of lymphatic vessel branching using the lymphatic endothelial cell marker LYVE one shown in red and neuro pill in two shown in green lymphatic vessels are visualized by both LYV one and NRP two, whereas LYV one is also expressed by a subset of macrophages indicated by the open arrow heads. This technique paves the way for researchers in the field of vascular biology to explore the complex patterning and the differentiation processes of vasculature during tissue development, tissue repairs, and in disease conditions.

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

Research

JoVE Journal

Biology

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

Bioengineering Human Microvascular Networks in Immunodeficient Mice

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

A Method for Labeling Vasculature in Embryonic Mice

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.

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

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

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.

Analysis of Coronary Vessels in Cleared Embryonic Hearts

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.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

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.

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

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.

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

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.

A Patient-Derived Xenograft Model for Venous Malformation

This assay utilizes mouse embryonic stem cells differentiated into embryoid bodies cultured in 3D-collagen gel to analyze the biological processes that control sprouting angiogenesis in vitro. The technique can be applied for testing drugs, modeling diseases, and for studying specific genes in the context of deletions that are embryonically lethal.

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

This protocol introduces a rapid method for quantitative whole-mount three-dimensional vascular imaging using light-sheet fluorescence microscopy. The efficacy of the method is demonstrated using the pharyngeal arch artery system of the chick embryo model, with hemodynamic forces quantified via computational fluid dynamics.

Rapid Whole-Mount High-Resolution Imaging of Small Animal Vasculature for Quantitative Studies

Learn whole-mount immunohistochemistry for vascular branching morphogenesis in mouse embryonic limb skin. This method enables high-resolution confocal imaging of blood and lymphatic vessel development.

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

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

Explore More Videos