This research was supported by the Brigham &#38; Women&#8217;s Hospital and the March of Dimes. The authors would like to thank all of the members of the Whited Lab for their support and advice.

All axolotl experimentation was performed in accordance with Brigham and Women&#39;s Hospital&#39;s (BWH) Institutional Animal Care and Use Committee.
1. Set up Perfusion Experiment
<ol>
	<li>Place an adult axolotl in a plastic container filled with 0.1% tricaine solution (MS222) for 15-20 min or until completely anesthetized. Ensure that the container is filled with enough tricaine solution such that the axolotl is completely submerged. 
	Note: All procedures must be performed in accor...

<ol>
	<li>Giuvarasteanu, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+electron+microscopy+of+vascular+corrosion+casts+-+standard+method+for+studying+microvessels.">Scanning electron microscopy of vascular corrosion casts - standard method for studying microvessels.</a> Rom J Morphol Embryo. 48 (3), 257-261 (2007).</li><li>Hasan, M. R., Herz, J., Hermann, D. M., Doeppner, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+perfusion+of+carbon+black+ink+allows+reliable+visualization+of+cerebral+vessels.">Intravascular perfusion of carbon black ink allows reliable visualization of cerebral vessels.</a> J Vis Exp. (71), e4374 (2013).</li><li> Minnich, B., Lametschwandtner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=+Scanning+electron+microscopy+and+vascular+corrosion+casting+for+the+characterization+of+microvascular+networks+in+human+and+animal+tissues."> Scanning electron microscopy and vascular corrosion casting for the characterization of microvascular networks in human and animal tissues.</a> Microscopy: Science, Technology, Applications, and Education. 1, 29-39 (2010).</li><li> Honig, M., Hume, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=+DiI+and+DiO:+versatile+fluorescent+dyes+for+neuronal+labelling+and+pathway+tracing."> DiI and DiO: versatile fluorescent dyes for neuronal labelling and pathway tracing.</a> Trends Neurosci. 13, 333-335 (1989).</li><li>Honig, M. G., Hume, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+carbocyanine+dyes+allow+living+neurons+of+identified+origin+to+be+studied+in+long-term+cultures.">Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in long-term cultures.</a> J Cell Biol. 103 (1), 171-187 (1986).</li><li>Schwartz, M., Agranoff, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outgrowth+and+maintenance+of+neurites+from+cultured+goldfish+retinal+ganglion+cells.">Outgrowth and maintenance of neurites from cultured goldfish retinal ganglion cells.</a> Brain Res. 206 (2), 331-343 (1981).</li><li>Li, Y., Song, Y., Zhao, L., Gaidosh, G., Laties, A. M., Wen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+labeling+and+visualization+of+blood+vessels+with+lipophilic+carbocyanine+dye+DiI.">Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI.</a> Nat Protoc. 3 (11), 1703-1708 (2008).</li><li>Kuo, T. H., Kowalko, J. E., DiTommaso, T., Nyambi, M., Montoro, D. T., Essner, J. J., Whited, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+TALEN-mediated+gene+editing+of+an+endogenous+locus+in+axolotl.">Evidence of TALEN-mediated gene editing of an endogenous locus in axolotl.</a> Regeneration. 2 (1), 37-43 (2015).</li><li>Brockes, J. P., Kumar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appendage+Regeneration+in+Adult+Vertebrates+and+Implications+for+Regenerative+Medicine.">Appendage Regeneration in Adult Vertebrates and Implications for Regenerative Medicine.</a> Science. 310 (5756), 1919-1923 (2005).</li><li>Smith, A. R., Wolpert, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerves+and+angiogenesis+in+amphibian+limb+regeneration.">Nerves and angiogenesis in amphibian limb regeneration.</a> Nature. 257 (5523), 224-225 (1975).</li></ol>

The authors have nothing to disclose.

Visualization of the vasculature of the axolotl can be successfully accomplished via perfusion with the lipophilic carbocyanine dye, DiI. In this study, we describe a novel protocol for the perfusion of the axolotl with DiI using a peristaltic pump. We also show the subsequent visualization of the axolotl vasculature using a fluorescent confocal microscope. This protocol was an adaptation of the rodent DiI perfusion protocol seen in Li et al.7, however major differences between the rodent...

Visualization of vasculature plays a vital role in understanding the structure and function of organisms across many species. Starting in the 16th century with Leonardo da Vinci, models and graphic representations of the circulation have been studied1. Using waxes and rubber molds, tissues were perfused to create three-dimensional models of the vasculature, which allowed for the study of organogenesis and pathogenesis1,2. Resins and waxes were colored with dyes such as India Ink or carmine red to allow for their easy visualization1,2. However, these techniques caused many issues because their high viscosities prevented full perfusion of the tissue of interest1. As the field became more sophisticated, the use of confocal and electron microscopes came into play, moving the perfusion techniques away from cast-molds and toward liquid perfusions of the vasculature, some of which allowed for the perfusion and imaging of blood vessels without destroying the initial tissue3. DiI, a fluorescent carbocyanine dye, is one such stain that allows for the perfusion of animals without damage to the vascular tissue.
Carbocyanine dyes are lipophilic dyes that incorporate into cell membranes upon contact. These dyes allow for easy and instantaneous staining of vascular endothelial cells, which can then be viewed under a fluorescent confocal microscope. DiI moves via lateral diffusion in the lipid membrane of cells, as shown in the labeling and tracing of neurons4. Chemically, the two alkyl chains of DiI give the dye its high affinity for cell membranes, while two conjugated rings from a fluorochrome which is responsible for emitting a red wavelength when excited by green fluorescent light filters4. DiI has been utilized in many capacities, including successful labeling of the plasma membrane and both anterograde and retrograde labeling in neurons5,6. DiI has previously been used in perfusion protocols while visualizing the vasculature of mice7.
Axolotls (Ambystoma mexicanum) are salamanders that live exclusively in brackish lakes near Mexico City, Mexico. These animals have become an important model for understanding regenerative processes as they can regenerate full limbs, tail (including nerve cord), portions of the heart and other internal organs, and portions of the eye as adults8,9. Additionally, with the recent application of genetic tools in axolotls, unprecedented insight into the molecules and cells driving these processes is now possible8. The successful regeneration of an entire limb requires an extensive revascularization process, which may play a significant role in regeneration beyond simply the traditional functions of blood vessels in providing oxygen and nutrients. Understanding revascularization in the context of tissue regeneration is imperative. Axolotl blood vessels have previously been visualized using India Ink, and while the results were intriguing, this process has not been revisited in subsequent decades10. We sought to adapt a DiI perfusion protocol developed for use in mammals to allow for a complete perfusion and visualization of the axolotl vasculature7. This protocol describes the steps taken to successfully perfuse and subsequently visualize the axolotl circulation with a DiI staining technique. This procedure will allow for precise visualization of patent blood vessels in homeostatic tissues, as well as in regenerating tissues, and provides a novel method for visualization and analysis of the revascularization process in the axolotl.

<table border="0" cellpadding="0" cellspacing="0" width="1583">
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:405px;">Peristaltic Pump&#160;</td>
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			<td height="42" style="height:42px;width:405px;">Perfusion tubing</td>
			<td style="width:195px;">Excelon Lab &#38; Vacuum Tubing</td>
			<td>436901705</td>
			<td>size S1A</td>
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			<td style="width:195px;">AmericanBio</td>
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			<td height="21" style="height:21px;">NaH2PO4</td>
			<td style="width:195px;">AmericanBio</td>
			<td>10049-21-5</td>
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			<td height="21" style="height:21px;">Distilled water</td>
			<td style="width:195px;"></td>
			<td style="width:152px;"></td>
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			<td height="24" style="height:24px;">1,1&#8242;-Dioctadecyl-3,3,3&#8242;,3&#8242;-tetramethylindocarbocyanine perchlorate</td>
			<td style="width:195px;">Sigma Aldrich</td>
			<td>468495</td>
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			<td style="width:195px;">Sigma Aldrich</td>
			<td>64-17-5</td>
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			<td>any polystyrene foam square with an axolotl-shaped&#160; cut out</td>
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			<td height="21" style="height:21px;width:405px;">Surgical scissors</td>
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			<td height="21" style="height:21px;width:405px;">Adult axolotl</td>
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			<td>S5761-500G</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Plastic tanks</td>
			<td style="width:195px;"></td>
			<td style="width:152px;"></td>
			<td>Varying size appropriate for the axolotl</td>
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			<td height="21" style="height:21px;width:405px;">Paraformaldehyde</td>
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			<td height="21" style="height:21px;width:405px;">Axolotl</td>
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			<td height="21" style="height:21px;width:405px;">Leica Microscope</td>
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			<td style="width:195px;">Leica</td>
			<td>M205FA/M165FC</td>
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		</tr>
	</tbody>
</table>

dii perfusion as a method for vascular visualization in ambystoma mexicanum

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that has emerged as an essential model for regeneration studies. Little is known about how revascularization occurs in the context of regeneration in these animals. Here we report a simple method for visualization of the vasculature in axolotl via perfusion of 1,1&#8217;-Dioctadecy-3,3,3&#8217;,3&#8217;-tetramethylindocarbocyanine perchlorate (DiI). DiI is a lipophilic carbocyanine dye that inserts into the plasma membrane of endothelial cells instantaneously. Perfusion is done using a peristaltic pump such that DiI enters the circulation through the aorta. During perfusion, dye flows through the axolotl&#8217;s blood vessels and incorporates into the lipid bilayer of vascular endothelial cells upon contact. The perfusion procedure takes approximately one hour for an eight-inch axolotl. Immediately after perfusion with DiI, the axolotl can be visualized with a confocal fluorescent microscope. The DiI emits light in the red-orange range when excited with a green fluorescent filter. This DiI perfusion procedure can be used to visualize the vascular structure of axolotls or to demonstrate patterns of revascularization in regenerating tissues.

With DiI staining, the vasculature of the axolotl can be easily visualized. Blood vessels of animals perfused with the lipophilic dye are immediately visible under a fluorescent confocal microscope. Figure 1.1-1.5 is a schematic representation of the perfusion protocol. After perfusion with the bright pink dye, a successfully perfused axolotl will appear pink. Using a green fluorescent filter on a confocal microscope a red emission of the vas...

Watch this Scientific Journal Video about DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum at JoVE.com

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Using a lipophilic 1,1&#39;-Dioctadecy-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate (DiI) staining technique, Ambystoma mexicanum can undergo vascular perfusion to allow for easy visualization of the vasculature.

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that ...

The overall goal of this procedure is to visualize the vasculature of the axolotl. This method can help answer key questions in the regenerative medicine field about the intricacies of the revascularization of regenerating tissues in the axolotl. The main advantage of this vascular visualization technique is that, all of the blood vessels in the animal can be easily imaged on a standard fluorescent microscope.Though this method can provide insight into axolotl limb regeneration, it can also be applied to other tissues, such as the tail, heart, eyes, gills or other organs. Before opening axolotl chest, first confirm the appropriate level of sedation by toe pinch. Then, use surgical forceps to pinch the skin along the central axis of the chest, just below the line of the shoulders.Using a scalpel, make a incision where the skin is pulled up and remove a square patch of skin over the chest to reveal two cartilage plates. Next, open a window over the thoracic cavity and carefully tear the connective tissue without cutting any major blood vessels. Use forceps to lift each cartilage plate individually, excising each plate with surgical scissors.Carefully pinch the exposed pericardium with the forceps. Pulling the tissue up and puncturing it with the surgical scissors, just deeply enough to remove the pericardium without injuring the heart. Then, gently remove the pericardium to expose the heart and aorta, to clearly view the heart at approximately five millimeters of the aorta, branching off of the heart.To perfuse the axolotl, place a clamp stand with a loaded butterfly needle next to a polystyrene foam frame, such that the arm of the clamp is easily manipulated for needle insertion. Turn on a peristaltic pump to flush 0.7x PBS through the pump tubing and point the needle tip toward the rostral aspect. Slide the forceps under the aortic arch and pull up slightly.Then, maneuver the needle clamp combination, so that the needle runs along the length of the aorta toward the head of the animal, and insert the needle about five millimeters into the aorta, keeping the forceps behind the aorta for support. A successful insertion can be confirmed by a visible enlargement of the heart atria. Quickly lacerate one atrium and allow the blood to drain.Then, perfuse the axolotl with about 20 to 30 milliliters of PBS. After a successful perfusion, the animal should change from light pink to white in color. When all of the PBS has been perfused, pause the peristaltic pump and move the free end of the tubing, into a 15 milliliter tube of Dil solution.Restart the pump, taking care to avoid creating bubbles in the tubing and perfuse the animal with the entire working Dil stock. In a successful perfusion, the axolotl will change to a bright pink color that will be most noticeable in the gills. When all of the Dil has been perfused, pause the pump and place the free end of the tubing into 4%paraformaldehyde solution.Then, restart the pump and perfuse at least 10 milliliters of the fixative. When all of the paraformaldehyde has been administered, stop the pump and remove the needle. Transfer the axolotl onto a plastic dish, and place the dish onto a fluorescent microscope stage.Then, with the lights off, select a filter that permits excitation of Dil, which emits a red orange light. After a successful perfusion, the Dil stain is visible as a red admission signal in all of the axolotl tissues, including the tail, limbs, gills and eyes. Once mastered, this technique can be completed in one hour if it is performed properly.While attempting this procedure, it's important to remember to take care not to lacerate the major blood vessels or the heart while opening the chest cavity and to insert the needle in line with the aorta to prevent through and through punctures. Following this procedure, other methods like, skeletal preparations can be performed to answer additional questions. After its development, this technique paved the way for researchers in the biomedical field to explore the vasculature of different species.After watching this video, you should have a good understanding of how to perform a cardiac perfusion of the axolotl with the carbocyanine dye, Dil. Don't forget, that working with needles and paraformaldehyde can be extremely hazardous and that precautions, such as wearing the appropriate personal protective equipment, should always be taken while performing the procedure.

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9.8K vistas.  Harvard Medical School and Brigham and Women's Hospital. The overall goal of this procedure is to visualize the vasculature of the axolotl. This method can help answer key questions in the regenerative medicine field about the intricacies of the revascularization of regenerating tissues in the axolotl. The main advantage of this vascular visualization technique is that, all of the blood vessels in the animal can be easily imaged on a standard fluorescent microscope.Though this method can provide insight into axolotl limb regeneration, it can...