The research activities of the authors are supported by grants from the National Institute of Health to Dr. Abe (HL-130193, HL-123346, HL-118462, HL-108551).

The protocols for the mouse partial carotid artery ligation and isolation of the mouse aorta and carotid artery for en face immunostaining are approved by the Institutional Animal Care and Use Committee (IBT 2014-9231).
1. Left Partial Carotid Artery Ligation
<ol>
	<li>Prepare the surgical space by placing a 12 inch x 14 inch heating pad on the table and cover the pad and table top with a large clean surgical drape. Adjust the arm of the boom stand so that the stereomicroscope&#39;s field of...

<ol>
	<li>Jelev, L., Surchev, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+simple+technique+for+en+face+endothelial+observations+using+water-soluble+media+-'thinned-wall'+preparations.">A novel simple technique for en face endothelial observations using water-soluble media -'thinned-wall' preparations.</a> J Anat. 212 (2), 192-197 (2008).</li><li>Neill, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+on+venous+endothelium+of+alterations+in+blood+flow+through+the+vessels+in+vein+walls,+and+the+possible+relation+to+thrombosis.">The effect on venous endothelium of alterations in blood flow through the vessels in vein walls, and the possible relation to thrombosis.</a> Ann Surg. 126 (3), 270-288 (1947).</li><li>Rogers, K. A., Kalnins, V. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+examining+the+endothelial+cytoskeleton+in+situ+using+immunofluorescence.">A method for examining the endothelial cytoskeleton in situ using immunofluorescence.</a> J Histochem Cytochem. 11 (11), 1317-1320 (1983).</li><li>Poole, J. C. F., Sanders, A. G., Florey, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regeneration+of+aortic+endothelium.">The regeneration of aortic endothelium.</a> J Pathol Bacteriol. 75, 133-143 (1958).</li><li>Sade, R. M., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=En+face+stripping+of+vascular+endothelium.">En face stripping of vascular endothelium.</a> Microvasc Res. 4, 77-80 (1972).</li><li>White, G. E., Gimbrone, M. A., Fujiwara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+influencing+the+expression+of+stress+fibers+in+vascular+endothelial+cells+in+situ.">Factors influencing the expression of stress fibers in vascular endothelial cells in situ.</a> J Cell Biol. 97 (2), 416-424 (1983).</li><li>Kim, D. W., Gotlieb, A. I., Langille, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+modulation+of+endothelial+F-actin+microfilaments+by+experimental+alterations+in+shear+stress.">In vivo modulation of endothelial F-actin microfilaments by experimental alterations in shear stress.</a> Arteriosclero. 9, 439-445 (1989).</li><li>Haka, A., Potteaux, S., Fraser, H., Randolph, G., Maxfield, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+monocyte+subpopulations+in+murine+atherosclerotic+plaques+by+multiphoton+microscopy.">Quantitative analysis of monocyte subpopulations in murine atherosclerotic plaques by multiphoton microscopy.</a> Plos ONE. 7 (9), 244823e (2012).</li><li>Ch&#232;vre, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Resolution+imaging+of+intravascular+atherogenic+inflammation+in+live+mice.">High-Resolution imaging of intravascular atherogenic inflammation in live mice.</a> Circ Res. 114, 770-779 (2014).</li><li>Heo, K. -. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disturbed+flow-activated+p90RSK+kinase+accelerates+atherosclerosis+by+inhibiting+SENP2+function.">Disturbed flow-activated p90RSK kinase accelerates atherosclerosis by inhibiting SENP2 function.</a> J Clin Invest. 125 (3), 1299-1310 (2015).</li><li>Le, N. -. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+arterial+blood+pressure+in+different+strains+of+mice.">Comparison of arterial blood pressure in different strains of mice.</a> Am J Hypertens. 14 (5), 405-408 (2001).</li><li>Jinguji, Y., Fujiwara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+fiber+dependent+axial+organization+of+fibronectin+fibrils+in+the+basal+lamina+of+the+chick+and+mesenteric+artery.">Stress fiber dependent axial organization of fibronectin fibrils in the basal lamina of the chick and mesenteric artery.</a> Endothelium. 2, 35-47 (1994).</li><li>Neckel, P. H., Mattheus, U., Hirt, B., Lust, L., Mack, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+tissue+clearing+(PACT):+Technical+evaluations+and+new+perspectives+in+immunofluorescence,+histology,+and+ultrastructure.">Large-scale tissue clearing (PACT): Technical evaluations and new perspectives in immunofluorescence, histology, and ultrastructure.</a> Sci Rep. 6, 34331 (2016).</li></ol>

When handling mouse blood vessels, it is important to remember that the endothelium is fragile and that any excessive mechanical force will damage endothelial cells. For example, endothelial cells break or detach from the vessel wall if the vessel is perfused too forcefully, which can easily happen when the vasculature is perfused using a hand-operated syringe.
To obtain constant perfusion pressure, we use a gravity perfusion system with a 120 cm water column pressure. It has been reported tha...

For histopathological studies by light microscopy, three dimensional pieces of biological tissues are routinely processed for paraffin embedment followed by sectioning and staining. A tissue sample that has been paraffin-embedded may be several millimeters in all three dimensions. However, for the purpose of light microscopy, it must be first sectioned so that light can pass through and then stained so that the thin section yields enough contrast for imaging. Because sectioned specimens are usually 5-10 &#181;m in thickness, one sees only a very small fraction of the whole specimen in two dimensions at a time. It is possible to collect sequential sections and, after imaging each section individually, perform computer-assisted reconstruction of the 3D images, but this is a tedious job indeed. Histopathology of blood vessels, especially for studying the pathogenesis of atherosclerosis, presents unique problems. Atherosclerosis is a focalized disease that develops locally in areas where disturbed blood flow occurs. Furthermore, the disease is initiated within the intima, a thin tissue consisting of a monolayer of endothelial cells and extracellular matrix, of large arteries. For these reasons, it is a challenge to locate and study early lesions using sectioned blood vessels because one can easily miss sectioning the lesion. Even if a section does include a diseased area, one will see only a 5-10 &#181;m portion containing endothelial cells and other vascular wall cells in the media and adventitia.
Whole mount en face (pronounced &#228;n &#712;f&#228;s) preparations allow us to survey a wide area of the blood vessel surface such as the entire aorta from the aortic root all the way down to the common iliac arteries. Using such a specimen stained with specific antibodies and other specific probes, one can pinpoint the location of lesions and also where various molecular events occur in endothelial cells in conjunction with atherogenesis such as changes in the expression, localization, and posttranslational modifications of proteins. In addition to studying atherogenesis, the endothelial cell shape observed in en face preparations is used as an indicator of the regional time-averaged blood flow pattern. Such data are important for studying mechanosignaling of endothelial cells in situ. For this purpose, routine histological cross-sectioned blood vessels are not useful. Thus, for vascular medicine and biology, it is especially important to acquire a technique for making en face preparations of blood vessels that allows one to observe a wide area of the vessel surface as well as the deeper subsurface areas of the vessel.
As reviewed by Jelev and Surchev1, vascular biologists have developed various methods to observe the lining of blood vessels en face. Some ingenious methods were developed in the 1940&#39;s and 1950&#39;s. Using these methods, they were able to study the fundamental organization of endothelial cells that line the inner surface of blood vessels. However, because of the way these en face preparations are prepared (the so-called Hautchen method2,3,4 or peeling off of the vessel surface5) and the way the specimen was stained, it was not always possible to obtain uninterrupted morphological information from the vessel surface into the deeper areas of the blood vessel wall. Whole mount en face vessel preparation combined with immunofluorescence staining allowed us to not only study endothelial cell morphology and protein expression and localization in these cells, but also to extend such studies to the subendothelial region of the vessel wall. Early studies using blood vessel en face preparations stained immunoflurescently began to appear in the 1980&#39;s6,7. With the advent of laser scanning confocal microscopy and more recently multiphoton microscopy, one can now obtain clear in-focus images of the blood vessel wall structure in immunofluorescently stained en face vessel samples as well as the vascular network in live animals8,9,10,11. These computer-based imaging techniques create in-focus optical sectioned images, and by stacking up such images, one can obtain reconstructed 3D images of the vessel wall and the vascular network in tissues. In addition, one can generate images of a section made along the Z-axis of the reconstructed image12,13.
In this article, we will illustrate a method for preparing en face preparations of the mouse aorta and the carotid artery for immunofluorescent staining. En face preparations can be made even after these vessels have been experimentally manipulated. For example, a carotid artery may be partially ligated and then an en face preparation made after such a surgery. For this reason, we will also describe in this article how we do a partial ligation on the carotid artery. Compared with making similar preparations from larger animals such as rats, rabbits, and humans, mouse vessels are small in size and more fragile, thus requiring added care for handling during surgical isolation of vessels and preparing them for antibody staining and microscopy. Because the most commonly used animal model for genetic modification is the mouse, it becomes critical for many investigators to handle mouse vessels without damaging them. In this manuscript, we will describe how to handle mouse blood vessels when making en face preparations of the mouse aorta and carotid artery. For the purpose of demonstration, we will use wild type C57/b6 mice.

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			<td>0.9% Sodium Chloride injection solution USP 500ml bag</td>
			<td>Fisher Scientific</td>
			<td>NC9788429</td>
		</tr>
		<tr>
			<td>12-well plates</td>
			<td>Fisher Scientific</td>
			<td>12556005</td>
		</tr>
		<tr>
			<td>6-0 coated vicryl suture</td>
			<td>Ethicon</td>
			<td>J833G</td>
		</tr>
		<tr>
			<td>AF488 goat anti-rat IgG&#160;</td>
			<td>Life Technologies</td>
			<td>A11006</td>
		</tr>
		<tr>
			<td>AF546 goat anti-rabbit IgG&#160;</td>
			<td>Life Technologies</td>
			<td>A11035</td>
		</tr>
		<tr>
			<td>Anti-CD144 (Ve-Cad)</td>
			<td>BD Biosciences</td>
			<td>BD555289</td>
		</tr>
		<tr>
			<td>Anti-VCAM-1 (H-276) Rabbit polyclonal IgG</td>
			<td>Santa Cruze Biotechnology</td>
			<td>Sc-8304</td>
		</tr>
		<tr>
			<td>Aoto Flow System</td>
			<td>Braintree Scientific</td>
			<td>EZ-AF9000</td>
		</tr>
		<tr>
			<td>Autoclave Wrap. 24x24in</td>
			<td>Cardinal Health</td>
			<td>4024</td>
		</tr>
		<tr>
			<td>Blunt retractors, 2.5mm wide</td>
			<td>Fine Science Tools</td>
			<td>18200-10</td>
		</tr>
		<tr>
			<td>Caprofen (Rimadyl)&#160;</td>
			<td>zoetis</td>
			<td>NADA#141-199</td>
		</tr>
		<tr>
			<td>Chlorhexidine Scrub, 2%</td>
			<td>Med-Vet International</td>
			<td>RXCHLOR2-PC</td>
		</tr>
		<tr>
			<td>Curity gauze sponges 2x2</td>
			<td>Cardinal Health</td>
			<td>KC2146</td>
		</tr>
		<tr>
			<td>Electric heating pad, 12X14</td>
			<td>Fisher Scientific</td>
			<td>NC0667724</td>
		</tr>
		<tr>
			<td>Extra Fine Graefe Forceps</td>
			<td>Fine Science Tools</td>
			<td>11152-10</td>
		</tr>
		<tr>
			<td>Iris Scissors</td>
			<td>Fine Science Tools</td>
			<td>14090-11</td>
		</tr>
		<tr>
			<td>Micro cover glass 22x50mm</td>
			<td>VWR</td>
			<td>48393059</td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-18</td>
		</tr>
		<tr>
			<td>normal goat serum</td>
			<td>Equitech-Bio</td>
			<td>GS05</td>
		</tr>
		<tr>
			<td>Paraformaldehyde Solution 4% in PBS</td>
			<td>Santa Cruze Biotechnology</td>
			<td>SC-281692</td>
		</tr>
		<tr>
			<td>Petri Dishes 100x15mm</td>
			<td>Fisher Scientific</td>
			<td>FB0875713</td>
		</tr>
		<tr>
			<td>Prolong Gold Antifade mountant with DAPI</td>
			<td>Life Technologies</td>
			<td>P-36935</td>
		</tr>
		<tr>
			<td>Puritan cortton swabs</td>
			<td>VWR</td>
			<td>10806-005</td>
		</tr>
		<tr>
			<td>Puritan Mini cotton tipped aplicators</td>
			<td>VWR</td>
			<td>82004-050</td>
		</tr>
		<tr>
			<td>Round handled Needle Holder</td>
			<td>Fine Science Tools</td>
			<td>12076-12</td>
		</tr>
		<tr>
			<td>Silk Suture 6/0</td>
			<td>Fine Science Tools</td>
			<td>18020-60</td>
		</tr>
		<tr>
			<td>Spring scissors</td>
			<td>ROBOZ</td>
			<td>RS-5601</td>
		</tr>
		<tr>
			<td>Strabismus Scissors</td>
			<td>Fine Science Tools</td>
			<td>14075-09</td>
		</tr>
		<tr>
			<td>Super Grip Forceps</td>
			<td>Fine Science Tools</td>
			<td>00649-11</td>
		</tr>
		<tr>
			<td>Transparent Dressing</td>
			<td>Cardinal Health</td>
			<td>TD-26C</td>
		</tr>
		<tr>
			<td>Triton X-1000</td>
			<td>Fisher Scientific</td>
			<td>AC327371000</td>
		</tr>
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en face preparation of mouse blood vessels

Sections of paraffin embedded tissues are routinely used for studying tissue histology and histopathology. However, it is difficult to determine what the three-dimensional tissue morphology is from such sections. In addition, the sections of tissues examined may not contain the region within the tissue that is necessary for the purpose of the ongoing study. This latter limitation hinders histopathological studies of blood vessels since vascular lesions develop in a focalized manner. This requires a method that enables us to survey a wide area of the blood vessel wall, from its surface to deeper regions. A whole mount en face preparation of blood vessels fulfills this requirement. In this article, we will demonstrate how to make en face preparations of the mouse aorta and carotid artery and to immunofluorescently stain them for confocal microscopy and other types of fluorescence-based imaging.

A typical en face immunofluorescence image of the endothelium is shown in Figure 3. This image shows a single optical section of a mouse aorta taken near the opening of an intercostal artery (the large dark egg-shaped area). The aorta was double-stained with anti-VE-cadherin (green) and anti-VCAM-1 (vascular cell adhesion molecule-1) (red). Each endothelial cell is outlined with a green linear staining at the adherens junction. Due to minor unevenness of the specimen, som...

Watch this Scientific Journal Video about En Face Preparation of Mouse Blood Vessels at JoVE.com

En Face Preparation of Mouse Blood Vessels

A procedure for making en face preparations of the mouse carotid artery and aorta is described. Such preparations, when immunofluorescently stained with specific antibodies, enable us to study localization of proteins and identification of cell types within the entire vascular wall by confocal microscopy.

En Face Preparation of Mouse Blood Vessels

Sections of paraffin embedded tissues are routinely used for studying tissue histology and histopathology. However, it is difficult to determine what the ...

The overall goal of this procedure is to prepare the mouse aorta and carotid artery for en face immuno staining after left carotid artery ligation. This method allows us to study organization, each protein expression, and possible state of antral cells and other vascular cells in immuno stain methods. En face preparations allow the analysis of large areas across blood vessels for the assessment of regional difference in the expression of various normal and possible cell parameters.Demonstrating the procedure will be Dr.Quaico a micro surgeon in our group. Begin by placing the anesthetized mouse onto a surgical board in the supine position. After confirming a lack of response to toe pinch tape the fore paws to the board in the out stretched position and both hind legs to the right side of the mouse to expose to left side of the neck.Remove the hair around the cervical area and disinfect the exposed skin. Cover the mouse with a sterilized surgical drape with a hole over the surgical region and apply ointment to the animal's eyes. Move the mouse under the dissecting microscope, and make a ventral mid line incision around the cervical area.Reposition the salivary glands that cover the blood vessels to the left side of the animal to expose the left common carotid artery, which bifurcates into the left internal carotid artery and the left external carotid artery. Gently remove the connective tissue around and below the left internal carotid artery and use a forceps to pass a 2.5 centimeter length of pre cut six O silk suture under the artery, using a second forceps to pull the suture roughly one third of its length to the other side of the vessel. Ligate the left internal carotid artery.Then remove the connective tissue around the left external carotid artery as just demonstrated, and make a ligation proximal to the left superior thyroid artery. When all of the arteries except the occipital artery have been ligated, return the salivary glands to their original positions and hydrate the surgical field with two to three drops of saline. Then use six O coated vicryl sutures to close the skin and place the mouse in the pre warmed cage with monitoring until full recumbency.At the termination of the experiment secure the mouse in the supine position on a dissecting board and use iris scissors to expose the abdominal cavity with a mid line incision. Cut the ribs laterally to the sternum to expose the thoracic cavity. Then nick the femoral artery and insert a 26 gauge needle attached to a gravity profusion set up into the apex of the left ventricle to profuse the circulatory system with saline solution supplemented with heparin.Continue the profusion until the saline flowing from the cut becomes clear. Then switch the profusion system from saline to 4%paraformaldehyde in PBS. After five minutes move the mouse under the dissecting microscope and use blunt end scissors and forceps to harvest the aorta and the left and right carotid arteries.Next, transfer the tissues into a Petri dish of PBS and carefully remove the fat and connective tissues, followed by the separation, and longitudinal splitting of the aorta and carotid arteries to expose the endothelium. The single most important thing to remember when making en face blood vessel preparations is that the endothelial cells are easily damaged by rough handling. Do not stretch the vessels at any time, especially during the harvesting and cleaning steps.Then transfer each vessel into individual wells of a 12 well plate containing 0.5 milliliters of permeabilizing solution per well. Permeabilize the blood vessels for 10 minutes with rocking at room temperature, followed by a brief wash in PBS. Block any nonspecific binding with a 30 minute incubation in 10%normal serum from the same species as the planned secondary antibodies in TTBS, with rocking at room temperature.Then label the samples with the primary antibodies of interest diluted in TTBS with 10%normal serum overnight with rocking at four degrees Celsius. The next morning, rinse the blood vessels with three 10 minute washes in TTBS with rocking at room temperature, followed by incubation in the appropriate secondary antibodies diluted in TTBS and 10%normal serum and dappy. Return the samples to the rocker and cover to protect from light.After one hour at room temperature with rocking wash the samples three times in fresh TTBS as demonstrated, followed by a brief rinse in PBS, and place one drop of anti fade reagent per vessel onto individual 22 by 50 millimeter cover glasses. Under a microscope, place one vessel into each drop of reagent with the endothelium facing down, and cover the vessels with one 22 by 75 millimeter slide glass per sample. Place the slides on a clean laboratory wipe and cover them with two pieces of laboratory wipe and 3.5 kilograms of weight to flatten the vessels.After a maximum of five minutes, remove the weight, and wipe the excess solution from around the cover slips. Then apply nail polish to the four corners of the cover slips and place the slides in a slide box, cover slip side up. The next morning use more nail polish to seal the cover slips completely and image the vessels as soon as the nail polish is dry.Here a typical en face immuno florescence image of endothelial double stained with anti VE cadherin and anti vascular cell adhesion molecule one antibodies and illustrating a single optical section of a mouse aorta taken near the opening of an intercostal artery is shown. Note the green linear outline staining at the adherence junction of the endothelial cells and the strong anti vascular cell adhesion molecule one staining at the opening of the intercostal artery where the disturbed blood flow is known to occur. In these images the en face staining of a partially ligated left carotid and a control unligated right artery one day after the surgery can be observed, with an increased anti vascular cell adhesion molecule one staining apparent in the ligated vessel.After watching this video you should have a good understanding of how to make a partial left carotid artery ligation and to make en face preparations in mouse blood vessels. Take this with your optical to other small animals. The use of en face preparations is now limited to immuno florescence staining, but can also be used with other methods, such as for study of those whole regions after oriole staining, or for ex vivo counter experiments.

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20.5K Views.  University of Texas MD Anderson Cancer Center. The overall goal of this procedure is to prepare the mouse aorta and carotid artery for en face immuno staining after left carotid artery ligation. This method allows us to study organization, each protein expression, and possible state of antral cells and other vascular cells in immuno stain methods. En face preparations allow the analysis of large areas across blood vessels for the assessment of regional difference in the expression of various normal and possible cell parameters.Demo...