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 view is in the center area of the heating pad.</li>
	<li>Turn on the heating pad on the table and set the 3-setting control dial to the medium heat level. At this temperature setting, the surgical board surface (see 1.6.1) will be 38-40 &#176;C.
	<ol>
		<li>Place a clean cage on another heating pad. Turn the heating pad on as above. This cage will be used for recovery after surgery (see 1.16) as well as housing.</li>
	</ol>
	</li>
	<li>On the surgical table, place an autoclaved sterilization pouch containing iris scissors (1 pair), tissue forceps (1 pair), super grip forceps (2 pairs), spring scissors (1 pair), blunt retractor (1 pair; 2.5 mm wide), round handled needle holder (1), sterilized 6-0 silk suture, cotton tipped applicators, mini cotton tipped applicators, surgical drapes, and 2&#34; x 2&#34; gauze sponges. Also place a squeeze bottle containing 70% ethanol and another containing chlorhexidine surgical scrubon the surgical table.</li>
	<li>Weigh a mouse. The body weight is needed to determine the appropriate amount of analgesia, which will be administered immediately before surgery.</li>
	<li>Place a mouse into the induction chamber.</li>
	<li>Turn on the oxygen tank and the anesthetic vaporizer in order to anesthetize the mouse in the induction chamber. Maintain the isoflurane level at 2%. It takes 3-5 min before the mouse stops moving.
	<ol>
		<li>While the mouse is being anesthetized, place a smaller piece of sterile surgical drape (24 inch x 24 inch) under the stereomicroscope to create a surgical surface. Then place an acrylic surgical board (which has been cleaned with 70% alcohol) on the draped surface. The surgical board should therefore be on the heating pad but separated by two layers of surgical drapes.</li>
	</ol>
	</li>
	<li>When the mouse stops moving in the induction chamber, transfer the mouse to a pre-surgical preparation area and position its nose into the nose-cone connected to the vaporizer (2% isoflurane). Remove the hair around the cervical area using an electric trimmer or hair remover lotion. We recommend hair removing lotion because this method will not produce loose hair pieces, which are difficult to completely remove from the surgical area.</li>
	<li>With the nose-cone in place, move the mouse to the surgical board.
	<ol>
		<li>Tape down the right and left fore-paws to the surgical board. Tape down both hind legs together on the right side of the mouse. This causes a slight rotation of the mouse body such that the left side of the neck area of the mouse becomes better positioned for surgery.</li>
	</ol>
	</li>
	<li>Disinfect the incision area with 70% alcohol, chlorhexidine surgical scrub, and again with 70% alcohol. Cover the mouse with a sterilized surgical drape except for the cervical incision area.</li>
	<li>Confirm by toe pinch that the mouse is fully anesthetized and give analgesia (Caprofen 3-5 mg/kg) via intraperitoneal or subcutaneous injection.</li>
	<li>Under the dissecting microscope, make a ventral midline incision around the cervical area using either a scalpel or iris scissors. 
	NOTE: We use scissors because the working distance of the stereomicroscope is limited, which makes it difficult to use a scalpel.</li>
	<li>Expose the left common carotid artery (LCCA) by pushing aside and repositioning the salivary glands that cover the blood vessels to the left side of the animal.</li>
	<li>Identify all the blood vessels in the surgical field (Figure 1). The LCCA bifurcates into the left internal carotid artery (ICA) and the left external carotid artery (ECA). The superficial thyroid artery (STA) arises from the ECA on the medial side. The occipital artery (OA) usually arises from the ECA, but in some mice it arises from the ICA.</li>
	<li>Ligate all artery branches except the OA using sterilized 6-0 silk suture. To achieve this, make the following two ligations.
	<ol>
		<li>Gently remove the connective tissue around and below the left internal carotid artery (ICA). Grab a piece of precut 6-0 silk suture (~2.5 cm) with forceps and pass it under the artery. Using another pair of forceps, pull the suture roughly 1/3 of its length and ligate the artery.</li>
		<li>Remove the connective tissue around the left external carotid artery (ECA) in the same way described above and make a ligation proximal to the left superior thyroid artery (STA) (Figure 1). Be careful not to damage nerve fibers that run within the surgical field.</li>
	</ol>
	</li>
	<li>When these ligations have been made, return the salivary glands to the original position and hydrate the surgical field by placing 2-3 drops of sterile saline. Close the skin using 6-0 coated vicryl sutures.</li>
	<li>After the surgery, place the mouse in the pre-warmed cage (see 1.2.1). The mouse should wake up within 5 min and begin to walk around. Once confirmed that the mouse behaves normally, bring the cage to the animal housing room.</li>
	<li>Observe the mouse daily for the first 3 days of recovery. The mouse can be kept as long as the experiment requires under the various conditions that the experimental protocol calls for. En face preparations may be made at any time after the surgery.</li>
</ol>
2. En Face Immunostaining
<ol>
	<li>Euthanize a mouse with CO2 by inhalation overdose.</li>
	<li>Tape the mouse in a supine (belly side up) position onto a dissecting board.</li>
	<li>Expose the abdominal cavity by making a midline incision using iris scissors.</li>
	<li>Expose the thoracic cavity by cutting the ribs laterally to the sternum.</li>
	<li>Make a nick in the vena cava or cut one of the femoral arteries for draining blood.</li>
	<li>Insert a 26 G needle attached to a gravity perfusion setup (120 cm water pressure) into the apex of the left ventricle and perfuse the circulatory system with saline solution containing heparin (40 U/mL). Continue the perfusion until the saline flowing out from the cut becomes clear.</li>
	<li>Switch the perfusion system from saline to the fixation solution containing 4% paraformaldehyde in PBS (phosphate buffered saline), and continue perfusing for 5 more min.</li>
	<li>Harvest the aorta and both left and right carotid arteries using blunt-end scissors and forceps, and place in a 50 mL conical tube containing the fixative on ice.</li>
	<li>Transfer the vessel into a Petri dish containing PBS under a dissecting microscope, and carefully remove the fat and connective tissues attached to the aorta and carotid arteries. Separate and split the aorta and carotid arteries longitudinally to expose the endothelium (Figure 2).</li>
	<li>Transfer each vessel separately to a well of a 12-well plate containing 0.5 mL of a permeabilizing solution (0.1% Triton X-100 in PBS) per well. Permeabilize blood vessels for 10 min with rocking at room temperature (RT).</li>
	<li>Wash briefly with PBS.</li>
	<li>To block non-specific antibody binding sites, incubate blood vessels in 10% normal serum from the animal species in which the secondary antibodies have been made, in TTBS (Tris-buffered saline (TBS) with 2.5% Tween 20) for 30 min with rocking at RT.</li>
	<li>Incubate vessels with the primary antibodies appropriately diluted in TTBS with 10% normal serum (as described above) overnight with rocking at 4 &#176;C. The level of dilution must be determined for each antibody.</li>
	<li>Perform the following control staining.
	<ol>
		<li>Incubate vessels with TTBS instead of a primary antibody followed by incubation with a secondary antibody.</li>
		<li>Incubate vessels with TTBS containing non-immune (or pre-immune) serum or Ig of the same animal (species) in which primary antibodies have been made, followed by incubation with a secondary antibody.</li>
		<li>Omit the incubation with a secondary antibody. These control specimens must be handled in the same manner and at the same time when staining with specific antibodies is performed.</li>
	</ol>
	</li>
	<li>Wash blood vessels 3 times with TTBS for 10 min each with rocking at RT.</li>
	<li>Incubate with fluorescently labeled secondary antibodies diluted appropriately in TTBS with 10% normal serum (as described above) for 1 h with rocking at RT. Nuclear staining with DAPI (4&#39;,6-diamidino-2-phenylindole) may be simultaneously performed at this stage by adding 1/5,000 by volume of a DAPI stock solution which contains 5 mg/mL of DAPI in H2O.</li>
	<li>Wash 3 times with TTBS for 10 min each with rocking at RT.</li>
	<li>Rinse briefly in PBS.</li>
	<li>Place one drop of the anti-fade reagent on a cover glass (22 mm x 50 mm) and place a blood vessel on the cover glass with the endothelium facing down.</li>
	<li>Place a slide glass (22 mm x 75 mm) on the blood vessel while avoiding trapping bubbles.</li>
	<li>Place the slide on a clean laboratory wipe (e.g. Kimwipe) and cover the slide with two pieces of laboratory wipe. Gently place 3.5 kg of weight (e.g. use a bottle of water on a thick book.) on the slide for a maximum of 5 min to flatten the en face blood vessel sample.</li>
	<li>Remove the weight and wipe off excess solution from around the coverslip.</li>
	<li>Apply nail polish at the 4 corners of the coverslip, place the slides in a slide box, coverslip side up, and keep in the dark at RT (or 4 &#176;C) overnight. This process flattens the tissue further and makes it easier to do microscopy at high magnifications.</li>
	<li>Seal the coverslip completely using nail polish.</li>
	<li>Perform microscopy as soon as the nail polish is dry.</li>
	<li>If needed, store slides at -20 &#176;C.</li>
</ol>

<ol><li>Jelev, L., Surchev, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+simple+technique+for+en+face+endothelial+observations+using+water-soluble+media+-%27thinned-wall%27+preparations%5BTitle%5D)+AND+%22J+Anat%22%5BJournal%5D)">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="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1803374/">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/pubmed?term=((A+method+for+examining+the+endothelial+cytoskeleton+in+situ+using+immunofluorescence%5BTitle%5D)+AND+%22J+Histochem+Cytochem%22%5BJournal%5D)">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="https://www.ncbi.nlm.nih.gov/pubmed/13576293">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/pubmed?term=((En+face+stripping+of+vascular+endothelium%5BTitle%5D)+AND+%22Microvasc+Res%22%5BJournal%5D)">En face stripping of vascular endothelium.</a> Microvasc Res. 4, 77-80 (1972).</li>
<li>White, G. E., Gimbrone, M. A. Jr, Fujiwara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Factors+influencing+the+expression+of+stress+fibers+in+vascular+endothelial+cells+in+situ%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">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/pubmed?term=((In+vivo+modulation+of+endothelial+F-actin+microfilaments+by+experimental+alterations+in+shear+stress%5BTitle%5D)+AND+%22Arteriosclero%22%5BJournal%5D)">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/pubmed?term=((Quantitative+analysis+of+monocyte+subpopulations+in+murine+atherosclerotic+plaques+by+multiphoton+microscopy%5BTitle%5D)+AND+%22Plos+ONE%22%5BJournal%5D)">Quantitative analysis of monocyte subpopulations in murine atherosclerotic plaques by multiphoton microscopy.</a> Plos ONE. 7 (9), 244823e(2012).</li>
<li>Chèvre, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-Resolution+imaging+of+intravascular+atherogenic+inflammation+in+live+mice%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">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/pubmed?term=((Disturbed+flow-activated+p90RSK+kinase+accelerates+atherosclerosis+by+inhibiting+SENP2+function%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">Disturbed flow-activated p90RSK kinase accelerates atherosclerosis by inhibiting SENP2 function.</a> J Clin Invest. 125 (3), 1299-1310 (2015).</li>
<li>Le, N. -T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+crucial+role+for+p90RSK-mediated+reduction+of+ERK5+transcriptional+activity+in+endothelial+dysfunction+and+atherosclerosis%5BTitle%5D)+AND+%22Circ%22%5BJournal%5D)">A crucial role for p90RSK-mediated reduction of ERK5 transcriptional activity in endothelial dysfunction and atherosclerosis.</a> Circ. 127, 486-499 (2013).</li>
<li>Kano, Y., Katoh, K., Masuda, M., Fujiwara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Macromolecular+composition+of+stress+fiber-plasma+membrane+attachment+sites+in+endothelial+cells+in+situ%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Macromolecular composition of stress fiber-plasma membrane attachment sites in endothelial cells in situ.</a> Circ Res. 79, 1000-1006 (1996).</li>
<li>Nigro, P., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/21173104">Cyclophilin A is an inflammatory mediator that promotes atherosclerosis in apolipoprotein E-dependent mice.</a> J Exp Med. 208 (1), 53-66 (2011).</li>
<li>Mattson, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+arterial+blood+pressure+in+different+strains+of+mice%5BTitle%5D)+AND+%22Am+J+Hypertens%22%5BJournal%5D)">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.tandfonline.com/doi/abs/10.3109/10623329409024632">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.nature.com/articles/srep34331">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.

<table><tbody><tr><td>0.9% Sodium Chloride injection solution USP 500 mL bag</td><td>Fisher Scientific</td><td>NC9788429</td><td></td></tr><tr><td>12-well plates</td><td>Fisher Scientific</td><td>12556005</td><td></td></tr><tr><td>6-0 coated vicryl suture</td><td>Ethicon</td><td>J833G</td><td></td></tr><tr><td>AF488 goat anti-rat IgG&amp;nbsp;</td><td>Life Technologies</td><td>A11006</td><td></td></tr><tr><td>AF546 goat anti-rabbit IgG&amp;nbsp;</td><td>Life Technologies</td><td>A11035</td><td></td></tr><tr><td>Anti-CD144 (Ve-Cad)</td><td>BD Biosciences</td><td>BD555289</td><td></td></tr><tr><td>Anti-VCAM-1 (H-276) Rabbit polyclonal IgG</td><td>Santa Cruze Biotechnology</td><td>Sc-8304</td><td></td></tr><tr><td>Aoto Flow System</td><td>Braintree Scientific</td><td>EZ-AF9000</td><td></td></tr><tr><td>Autoclave Wrap. 24 inch x 24 inch</td><td>Cardinal Health</td><td>4024</td><td></td></tr><tr><td>Blunt retractors, 2.5 mm wide</td><td>Fine Science Tools</td><td>18200-10</td><td></td></tr><tr><td>Caprofen (Rimadyl)&amp;nbsp;</td><td>zoetis</td><td>NADA#141-199</td><td></td></tr><tr><td>Chlorhexidine Scrub, 2%</td><td>Med-Vet International</td><td>RXCHLOR2-PC</td><td></td></tr><tr><td>Curity gauze sponges 2 x 2</td><td>Cardinal Health</td><td>KC2146</td><td></td></tr><tr><td>Electric heating pad, 12 x 14</td><td>Fisher Scientific</td><td>NC0667724</td><td></td></tr><tr><td>Extra Fine Graefe Forceps</td><td>Fine Science Tools</td><td>11152-10</td><td></td></tr><tr><td>Iris Scissors</td><td>Fine Science Tools</td><td>14090-11</td><td></td></tr><tr><td>Micro cover glass 22 mm x 50 mm</td><td>VWR</td><td>48393059</td><td></td></tr><tr><td>Microscope Slides</td><td>Fisher Scientific</td><td>12-550-18</td><td></td></tr><tr><td>normal goat serum</td><td>Equitech-Bio</td><td>GS05</td><td></td></tr><tr><td>Paraformaldehyde Solution 4% in PBS</td><td>Santa Cruze Biotechnology</td><td>SC-281692</td><td></td></tr><tr><td>Petri dishes 100 mm x 15 mm</td><td>Fisher Scientific</td><td>FB0875713</td><td></td></tr><tr><td>Prolong Gold Antifade mountant with DAPI</td><td>Life Technologies</td><td>P-36935</td><td></td></tr><tr><td>Puritan cortton swabs</td><td>VWR</td><td>10806-005</td><td></td></tr><tr><td>Puritan Mini cotton tipped aplicators</td><td>VWR</td><td>82004-050</td><td></td></tr><tr><td>Round handled Needle Holder</td><td>Fine Science Tools</td><td>12076-12</td><td></td></tr><tr><td>Silk Suture 6/0</td><td>Fine Science Tools</td><td>18020-60</td><td></td></tr><tr><td>Spring scissors</td><td>ROBOZ</td><td>RS-5601</td><td></td></tr><tr><td>Strabismus Scissors</td><td>Fine Science Tools</td><td>14075-09</td><td></td></tr><tr><td>Super Grip Forceps</td><td>Fine Science Tools</td><td>00649-11</td><td></td></tr><tr><td>Transparent Dressing</td><td>Cardinal Health</td><td>TD-26C</td><td></td></tr><tr><td>Triton X-1000</td><td>Fisher Scientific</td><td>AC327371000</td><td></td></tr></tbody></table>

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 ...

en-face-preparation-of-mouse-blood-vessels

Research

JoVE Journal

Biology

20.9K Views.  University of Texas MD Anderson Cancer Center.  Een procedure voor het maken van een gezichtspreparatie van de muizencarotisarterie en aorta wordt beschreven. Dergelijke preparaten, wanneer immunofluorescent gekleurd met specifieke antilichamen, ons in staat stellen om lokalisatie van eiwitten en identificatie van celtypen in de gehele vaatwand door confocale microscopie te bestuderen. 

Video: En Face Preparation of Mouse Blood Vessels

Murine Spinotrapezius Model to Assess the Impact of Arteriolar Ligation on Microvascular Function and Remodeling

The murine spinotrapezius is a thin, superficial skeletal support muscle that extends from T3 to L4, and is easily accessible via dorsal skin incision. Its unique anatomy makes the spinotrapezius useful for investigation of ischemic injury and subsequent microvascular remodeling. Here, we demonstrate an arteriolar ligation model in the murine spinotrapezius muscle that was developed by our research team and previously published1-3. For certain vulnerable mouse strains, such as the Balb/c mouse, this ligation surgery reliably creates skeletal muscle ischemia and serves as a platform for investigating therapies that stimulate revascularization. Methods of assessment are also demonstrated, including the use of intravital and confocal microscopy. The spinotrapezius is well suited to such imaging studies due to its accessibility (superficial dorsal anatomy) and relative thinness (60-200 &mu;m). The spinotrapezius muscle can be mounted en face, facilitating imaging of whole-muscle microvascular networks without histological sectioning. We describe the use of intravital microscopy to acquire metrics following a functional vasodilation procedure; specifically, the increase in arterilar diameter as a result of muscle contraction. We also demonstrate the procedures for harvesting and fixing the tissues, a necessary precursor to immunostaining studies and the use of confocal microscopy.

We demonstrate a novel arterial ligation model in murine spinotrapezius muscle, including a step-by-step procedure and description of required instrumentation. We describe the surgery and relevant outcome measurements relating to vascular network remodeling and functional vasodilation using intravital and confocal microscopy.

The murine spinotrapezius is a thin, superficial skeletal support muscle that extends from T3 to L4, and is easily accessible via dorsal skin incision. ...

Medicine

Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ability to switch cells from one pathway to another is highly attractive as a therapeutic target. We designed a decoy peptide IRF5D with a molecular modeling software for designing small molecules and peptides.
IRF5D inhibited IRF5, reduced alterations in extracellular matrix, and improved endothelial vasodilation in the tight-skin mouse (Tsk/+). The Kd of IRF5D for recombinant IRF5 is 3.72 &plusmn; 0.74 x 10-6 M as determined by binding experiments using biolayer interferometry experiments. Endothelial cells (EC) proliferation and apoptosis were unchanged using increasing concentrations of IRF5D (0 to 100 &#181;g/mL, 24 h). Tsk/+ mice were treated with IRF5D (1 mg/kg/d subcutaneously, 21 d). IRF5 and ICAM expressions were decreased after IRF5D treatment. Endothelial function was improved as assessed by vasodilation of facialis arteries from Tsk/+ mice treated with IRF5D compared to Tsk/+ mice without IRF5D treatment. As a transcription factor, IRF5 traffics from the cytosol to the nucleus. Translocation was assessed by immunohistochemistry on cardiac myocytes cultured on the different cardiac extracellular matrices. IRF5D treatment of the Tsk/+ mouse resulted in a reduced number of IRF5 positive nuclei in comparison to the animals without IRF5D treatment (50 &#181;g/mL, 24 h). These findings demonstrate the important role that IRF5 plays in inflammation and fibrosis in Tsk/+ mice.

We describe the isolation of cardiac extracellular matrix from C57Bl/6J control mice, tight-skin mice, and tight-skin mice treated with the IRF5 inhibitory peptide. We also describe the vasodilation studies on the isolated vessels from C57Bl/6J, tight-skin mice and tight-skin mice treated with the IRF5 inhibitory peptide.

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ...

Immunology and Infection

Purification of Mouse Brain Vessels

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson&#8217;s disease, Alzheimer&#8217;s disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified brain vessels.

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

Neuroscience

Retinal Pericytes Visualization Using Tissue-Clearing and Fluorescent Labeling

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining 

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal vasculature. To provide a detailed view of the morphological characteristics of pericytes, this study described a new approach combining the retro-orbital injection of a fluorescent agent, immunofluorescent-staining, and tissue-clearing treatment. Firstly, the fluorescent tomato lectin was injected into the retro-orbital sinus of the live mouse to label the retinal vasculature. After 5 min, the mouse was sacrificed, and its intact retina was carefully removed from the retinal cup and immunofluorescently stained with platelet-derived growth factor receptor &#946; to reveal the pericytes. Additionally, the stained retina was treated with tissue-clearing reagent and whole mounted on the microscope slide. Through these approaches, the retinal vasculature and pericytes were clearly observed in the transparent retina. Under a confocal microscope, we obtained more opportunities to take high-resolution images for further reconstructing and analyzing the morphological characteristics of pericytes along the retinal vascular tree in a three-dimensional view. Methodologically, this protocol offers an effective approach for visualizing pericytes within the retinal vasculature, providing valuable insights into their role under both physiological and pathological conditions.

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining

The pericytes in retinal vasculature were examined by immunofluorescent staining with platelet-derived growth factor receptor &#946; after retro-orbital injection of fluorescent tomato lectin. The labeled retina was further treated with the tissue-clearing method and whole mounted for visualizing the three-dimensional views of pericytes surrounding retinal vasculature under a confocal microscope.

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal ...

Biochemistry

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular diseases, such as diabetic retinopathy, hypertensive retinopathy, and possibly glaucoma. Therefore, retinal microvascular preparations are pivotal tools for physiological and pharmacological studies to delineate the underlying pathophysiological mechanisms and to design therapies for the diseases. Despite the wide use of mouse models in ophthalmic research, studies on retinal vascular reactivity are scarce in this species. A major reason for this discrepancy is the challenging isolation procedures owing to the small size of these retinal blood vessels, which is ~ &#8804; 30 &#181;m in luminal diameter. To circumvent the problem of direct isolation of these retinal microvessels for functional studies, we established an isolation and preparation technique that enables ex vivo studies of mouse retinal vasoactivity under near-physiological conditions. Although the present experimental preparations will specifically refer to the mouse retinal arterioles, this methodology can readily be employed to microvessels from rats.

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Many vision-threatening ocular diseases are associated with dysfunctional retinal microvessels. Therefore, the measurement of retinal arteriole responses is important to investigate the underlying pathophysiological mechanisms. This article describes a detailed protocol for mouse retinal arteriole isolation and preparation to assess the effects of vasoactive substances on vascular diameter.

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.

Here, a critical hindlimb ischemia experimental model is presented followed by a battery of functional, histologic and molecular tests&#160;to assess the effectiveness of angiogenic therapies.&#8203;

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard ...

Dolichoectasia: Vascular Dementia and Alzheimer&#39;s Disease Models

A Visual Approach for Inducing Dolichoectasia in Mice to Model Large Vessel-Mediated Cerebrovascular Dysfunction

The blood-brain (BBB) is a crucial system that regulates selective brain circulation with the periphery, as an example, allowing necessary nutrients to enter and expel excessive amino acids or toxins from the brain. To model how the BBB can be compromised in diseases like vascular dementia (VaD) or Alzheimer's disease (AD), researchers developed novel methods to model vessel dilatation. A compromised BBB in these disease states can be detrimental and result in the dysregulation of the BBB leading to untoward and pathological consequences impacting brain function. We were able to modify an existing technique that enabled us to inject directly into the Cisterna magna (CM) to induce dilatation of blood vessels using elastase, and disrupt the tight junctions (TJ) of the BBB. With this method, we were able to see various metrics of success over previous techniques, including consistent blood vessel dilatation, reduced mortality or improved recovery, and improving the fill/opacifying agent, a silicone rubber compound, delivery for labeling blood vessels for dilatation analysis. This modified minimally invasive method has had promising results, with a 19%-32% increase in sustained dilatation of large blood vessels in mice from 2 weeks to 3 months post-injection. This improvement contrasts with previous studies, which showed increased dilatation only at the 2 week mark. Additional data suggests sustained expansion even after 9.5 months. This increase was confirmed by comparing the diameter of blood vessels of the elastase and the vehicle-injected group. Overall, this technique is valuable for studying pathological disorders that affect the central nervous system (CNS) using animal models.

Author Spotlight: Modeling Vascular Contributions to Alzheimer's Disease in Transgenic Mice

We demonstrate chemically inducing large blood vessel dilatation in mice as a model for investigating cerebrovascular dysfunction, which can be used for vascular dementia and Alzheimer's disease modeling. We also demonstrate visualizing the vasculature by injecting silicone rubber compound and providing clear visual guidance for measuring changes in blood vessel size.

The blood-brain (BBB) is a crucial system that regulates selective brain circulation with the periphery, as an example, allowing necessary nutrients to ...

Using En Face Immunofluorescence Staining to Observe Vascular Endothelial Cells Directly

Aberrant changes in endothelial phenotype and morphology are considered to be initial events in the pathogenesis of atherosclerosis. Direct observation of the intact endothelium will provide valuable information for understanding the cellular and molecular events in the dysfunctional endothelial cells. Here, we describe a modified en face immunofluorescence staining technique which enables scientists to obtain clear images of the intact endothelial surface and analyze the molecule expression patterns in situ. The method is simple and reliable for observing the entire endothelial monolayer at different sites of the aorta. This technique may be a promising tool for understanding the pathophysiology of atherosclerosis, especially at an early stage.

Here, we present a protocol for immunofluorescence staining to observe the endothelial cells of the mouse aorta directly. This technique is useful when studying the cellular and molecular phenotype of endothelial cells in different flow patterns and in the development of atherosclerosis.

Aberrant changes in endothelial phenotype and morphology are considered to be initial events in the pathogenesis of atherosclerosis. Direct observation of ...

Puncture Wound Hemostasis and Montaged Electron Microscopy 

Puncture Wound Hemostasis and Preparation of Samples for Montaged Wide-Area Electron Microscopy Analysis

Hemostasis, the process of normal physiological control of vascular damage, is fundamental to human life. We all suffer minor cuts and puncture wounds from time to time. In hemostasis, self-limiting platelet aggregation leads to the formation of a structured thrombus in which bleeding cessation comes from capping the hole from the outside. Detailed characterization of this structure could lead to distinctions between hemostasis and thrombosis, a case of excessive platelet aggregation leading to occlusive clotting. An imaging-based approach to puncture wound thrombus structure is presented here that draws upon the ability of thin-section electron microscopy to visualize the interior of hemostatic thrombi. The most basic step in any imaging-based experimental protocol is good sample preparation. The protocol provides detailed procedures for preparing puncture wounds and platelet-rich thrombi in mice for subsequent electron microscopy. A detailed procedure is given for in situ fixation of the forming puncture wound thrombus and its subsequent processing for staining and embedding for electron microscopy. Electron microscopy is presented as the end imaging technique because of its ability, when combined with sequential sectioning, to visualize the details of the thrombus interior at high resolution. As an imaging method, electron microscopy gives unbiased sampling and an experimental output that scales from nanometer to millimeters in 2 or 3 dimensions. Appropriate freeware electron microscopy software is cited that will support wide-area electron microscopy in which hundreds of frames can be blended to give nanometer-scale imaging of entire puncture wound thrombi cross-sections. Hence, any subregion of the image file can be placed easily into the context of the full cross-section.

Author Spotlight: Revealing Platelet Dynamics Through Advances in Structural Hematology

A puncture wound procedure for hemostatic thrombus formation is presented here. The formed thrombi are large and are hundreds of microns in diameter. Hence, volume imaging approaches are appropriate. We suggest montaged wide-area transmission electron microscopy as a high-resolution approach available to many and detail a preparative protocol.

Hemostasis, the process of normal physiological control of vascular damage, is fundamental to human life. We all suffer minor cuts and puncture wounds ...

Imaging Fixture Supported Mouse Liver Staging for Multiphoton Microscopy Based Vascular Visualization: A Stabilization Device-assisted Imaging Technique to Obtain Multi-dimensional Images of Blood Vessels in Mouse Liver

Source: Rongrong, W. et al. <a href="https://www.jove.com/video/60932" target="_blank">Multiphoton Microscopic Observation of Vessels in Mouse Liver Tissue</a>. J. Vis. Exp. (2021)
This video describes an imaging procedure to observe and study intravascular dynamics of a mouse liver tissue using multiphoton microscopy following stabilization of the organ. Multiphoton microscopy enables the imaging of biological tissue at high spatial resolution in vivo.

Source: Rongrong, W. et al. Multiphoton Microscopic Observation of Vessels in Mouse Liver Tissue. J. Vis. Exp. (2021)
This video describes an imaging ...

En Face Preparation of Mouse Blood Vessels

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