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 ...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
			<div>
			Antibodies
Pan-endothelial cell marker
<table border="1">
 <tbody>
 <tr>
 <td>Antibody</td>
 <td>Species</td>
 <td>Company</td>
 <td>Catalogue # </td>
 <td>Working condition</td>
 </tr>
 <tr>
 <td>PECAM-1</td>
 <td>Armenian hamster (M)</td>
 <td>Chemicon</td>
 <td>MAB1398Z</td>
 <td>1:100 dilution#1</td>
 </tr>
 <tr>
 <td>PECAM-1</td>
 <td>Rat (M)</td>
 <td>BD</td>
 <td>553369</td>
 <td>1:300 dilution</td>
 </tr>
 <tr>
 <td>VEGFR2</td>
 <td>Rat (M)</td>
 <td>eBioscience</td>
 <td>14-5821-82</td>
 <td>1:200 dilution</td>
 </tr>
 <tr>
 <td>CD34</td>
 <td>Rat (M)</td>
 <td>eBioscience</td>
 <td>13-0341</td>
 <td>1:300 dilution</td>
 </tr>
 <tr>
 <td>Collagen IV</td>
 <td>Rabbit (P)</td>
 <td>AbD Serotec</td>
 <td>2150-1470</td>
 <td>1:300 dilution#2</td>
 </tr>
 </tbody>
</table>
Arterial endothelial cell marker
<table border="1">
 <tbody>
 <tr>
 <td>Antibody</td>
 <td>Species</td>
 <td>Company</td>
 <td>Catalogue # </td>
 <td>Working condition</td>
 </tr>
 <tr>
 <td>Neuropilin1</td>
 <td>Rabbit (P)</td>
 <td>The Alex Kolodkin's lab#3</td>
 <td></td>
 <td>1:3000 dilution</td>
 </tr>
 <tr>
 <td>Unc5H2</td>
 <td>Goat (P) </td>
 <td>R&amp;D</td>
 <td>AF1006</td>
 <td>1:200 dilution</td>
 </tr>
 </tbody>
</table>
Venous endothelial cell marker
<table border="1">
 <tbody>
 <tr>
 <td>Antibody</td>
 <td>Species</td>
 <td>Company</td>
 <td>Catalogue # </td>
 <td>Working condition</td>
 </tr>
 <tr>
 <td>EphB4</td>
 <td>Goat (P)</td>
 <td>R&amp;D</td>
 <td>AF446</td>
 <td>1:100 dilution</td>
 </tr>
 </tbody>
</table>
Lymphatic endothelial cell marker
<table border="1">
 <tbody>
 <tr>
 <td>Antibody</td>
 <td>Species</td>
 <td>Company</td>
 <td>Catalogue # </td>
 <td>Working condition</td>
 </tr>
 <tr>
 <td>LYVE-1#4</td>
 <td>Rabbit (P) &nbsp;</td>
 <td>Abcam</td>
 <td>ab14917</td>
 <td>1:200 dilution</td>
 </tr>
 <tr>
 <td>LYVE-1#4</td>
 <td>Rat (M) &nbsp;</td>
 <td>MBL</td>
 <td>D225-3</td>
 <td>1:200 dilution</td>
 </tr>
 <tr>
 <td>Prox-1</td>
 <td>Rabbit (P)</td>
 <td>Chemicon</td>
 <td>AB5475</td>
 <td>1:1000 dilution</td>
 </tr>
 <tr>
 <td>Prox-1</td>
 <td>Goat (P)</td>
 <td>R&amp;D</td>
 <td>AF2727</td>
 <td>1:50 dilution</td>
 </tr>
 <tr>
 <td>Neuropilin2</td>
 <td>Rabbit (P)</td>
 <td>Cell Signaling</td>
 <td>3366</td>
 <td>1;100 dilution</td>
 </tr>
 <tr>
 <td>Podoplanin</td>
 <td>Syrian hamster (M)</td>
 <td>Hybridoma Bank</td>
 <td>8.1.1</td>
 <td>1:200 dilution</td>
 </tr>
 </tbody>
</table>
Smooth muscle cell/pericyte marker
<table border="1">
 <tbody>
 <tr>
 <td>Antibody</td>
 <td>Species</td>
 <td>Company</td>
 <td>Catalogue # </td>
 <td>Working condition</td>
 </tr>
 <tr>
 <td>&alpha;SMA-Cy3</td>
 <td>Mouse (M)#5</td>
 <td>Sigma</td>
 <td>c-6198</td>
 <td>1:500 dilution#6</td>
 </tr>
 <tr>
 <td>NG2</td>
 <td>Rabbit (P) &nbsp;</td>
 <td>Chemicon</td>
 <td>AB5320</td>
 <td>1:200 dilution</td>
 </tr>
 <tr>
 <td>SM22&alpha;</td>
 <td>Rabbit (P)</td>
 <td>Abcam</td>
 <td>ab14106</td>
 <td>1:200 dilution</td>
 </tr>
 </tbody>
</table>
Antibodies forGFP reporter
<table border="1">
 <tbody>
 <tr>
 <td>Antibody</td>
 <td>Species</td>
 <td>Company</td>
 <td>Catalogue # </td>
 <td>Working condition</td>
 </tr>
 <tr>
 <td>GFP</td>
 <td>Rabbit (P) &nbsp;</td>
 <td>Invitrogen</td>
 <td>A11122</td>
 <td>1:300 dilution</td>
 </tr>
 <tr>
 <td>GFP</td>
 <td>Rat (M) &nbsp;</td>
 <td>Nacalai tesque</td>
 <td>04404-84</td>
 <td>1:1000 dilution</td>
 </tr>
 <tr>
 <td>GFP</td>
 <td>Chick (P)</td>
 <td>Chemicon</td>
 <td>P42212</td>
 <td>1:300 dilution</td>
 </tr>
 </tbody>
</table>
Antibodies for LacZ reporter
<table border="1">
 <tbody>
 <tr>
 <td>Antibody</td>
 <td>Species</td>
 <td>Company</td>
 <td>Catalogue # </td>
 <td>Working condition</td>
 </tr>
 <tr>
 <td>&beta;-gal</td>
 <td>Rabbit (P) &nbsp;</td>
 <td>MP Biomedical</td>
 <td>55976</td>
 <td>1:5000 dilution</td>
 </tr>
 <tr>
 <td>&beta;-gal</td>
 <td>Goat (P) </td>
 <td>AbD Serotec</td>
 <td>4600-1409</td>
 <td>1:500 dilution</td>
 </tr>
 <tr>
 <td>&beta;-gal</td>
 <td>Chick (P)</td>
 <td>Abcam</td>
 <td>ab9361</td>
 <td>1:200 dilution</td>
 </tr>
 </tbody>
</table>
Antibodies for peripheral axon
<table border="1">
 <tbody>
 <tr>
 <td>Antibody</td>
 <td>Species</td>
 <td>Company</td>
 <td>Catalogue # </td>
 <td>Working condition</td>
 </tr>
 <tr>
 <td>2H3</td>
 <td>Mouse (M)#7</td>
 <td>Hybridoma Bank</td>
 <td>2H3</td>
 <td>1:200 dilution</td>
 </tr>
 <tr>
 <td>Tuj1</td>
 <td>Mouse (M)#8 &nbsp;</td>
 <td>Covance</td>
 <td>MMS-435P</td>
 <td>1:500 dilution</td>
 </tr>
 <tr>
 <td>Peripherin</td>
 <td>Rabbit (P)</td>
 <td>Chemicon</td>
 <td>AB1530</td>
 <td>1:1000 dilution</td>
 </tr>
 </tbody>
</table>
Antibodies for migrating Schwann cells
<table border="1">
 <tbody>
 <tr>
 <td>BFABP</td>
 <td>Rabbit (P)</td>
 <td>The Thomas M&uuml;ller's lab#9</td>
 <td></td>
 <td>1:3000 dilution</td>
 </tr>
 </tbody>
</table>
(P): polyclonal antibody, (M): monoclonal antibody
#1: Goat anti-Armenian hamster-Cy3 (Jackson ImmunoResearch 127-165-160) antibody should be used as a secondary antibody.
#2: The Collagen IV antibody can be used to detect blood vessels after in situ hybridization.
#3: The Neuropilin1 antibody is kindly provided by the Alex Kolodkin's lab in the Johns Hopkins University. Sheep anti-human Neuropilin1 antibody is available in R&amp;D (AF3870), although we have not tested it yet.
#4: The LYVE-1 antibodies also detect a subset of macrophages in the embryonic skin.
#5: The anti-&alpha;SMA antibody is mouse IgG2a monoclonal antibody.
#6: The Cy3-conjugated &alpha;SMA antibody is incubated for 1 hour at room temperature together with secondary antibodies for other primary antibodies.
#7: 2H3 antibody is mouse IgG1 monoclonal antibody against neurofilament.
#8: Tuj1 antibody is mouse IgG2a monoclonal antibody against Neuron-specific class III beta-tubulin&#65294;
#9: The BFABP (brain-specific fatty acid binding protein) antibody is kindly provided by the Thomas M&uuml;ller's lab in Max-Delbr&uuml;ck-Center for Molecular Medicine.		</div>

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

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

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

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

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

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

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. Introduciamo un tutto-mount immunoistochimica e microscopia confocale a scansione laser con l'etichettatura per analizzare più intricata la formazione della rete vascolare nella pelle degli arti embrionali di topo.

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

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...