This study was supported by the National Natural Science Foundation of China (Grant No. 81670451, 81770430), the Shanghai Rising-Star Program (Grant No. 17QA1403000), and the Science Technology Committee of the Shanghai Municipal Government (Grant No. 14441903002, 15411963700).

All animal experiments were conducted in accordance with experimental protocols approved by the Committee on Animal Resources of Shanghai Jiao Tong University.
1. Perfusion of the mouse aorta
<ol>
	<li>Briefly, anesthetize 12-week-old C57BL/6 mice with intraperitoneal injections of sodium pentobarbital (50 mg/kg body weight). Confirm proper anesthetization by gently pinching the tail. 
	NOTE: If no movement is observed, the animal is sufficiently anesthetized to start the experiments.</...

<ol>
	<li>Gimbrone, M. A., Garcia-Cardena, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+Cell+Dysfunction+and+the+Pathobiology+of+Atherosclerosis.">Endothelial Cell Dysfunction and the Pathobiology of Atherosclerosis.</a> Circulation Research. 118 (4), 620-636 (2016).</li><li>Zhou, J., Li, Y. S., Chien, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shear+stress-initiated+signaling+and+its+regulation+of+endothelial+function.">Shear stress-initiated signaling and its regulation of endothelial function.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 34 (10), 2191-2198 (2014).</li><li>Tarbell, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shear+stress+and+the+endothelial+transport+barrier.">Shear stress and the endothelial transport barrier.</a> Cardiovascular Research. 87 (2), 320-330 (2010).</li><li>Warren, B. A. <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+the+production+of+&amp;#34;en+face&amp;#34;+preparations+one+cell+in+thickness.">A method for the production of &amp;#34;en face&amp;#34; preparations one cell in thickness.</a> Journal of Microscopy. 85 (4), 407-413 (1965).</li><li>Azuma, K., 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=A+new+En+face+method+is+useful+to+quantitate+endothelial+damage+in+vivo.">A new En face method is useful to quantitate endothelial damage in vivo.</a> Biochemical and Biophysical Research Communications. 309 (2), 384-390 (2003).</li><li>Son, D. J., 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=The+atypical+mechanosensitive+microRNA-712+derived+from+pre-ribosomal+RNA+induces+endothelial+inflammation+and+atherosclerosis.">The atypical mechanosensitive microRNA-712 derived from pre-ribosomal RNA induces endothelial inflammation and atherosclerosis.</a> Nature Communications. 4, 3000 (2013).</li><li>Go, Y. M., 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+enhances+inflammatory+signaling+and+atherogenesis+by+increasing+thioredoxin-1+level+in+endothelial+cell+nuclei.">Disturbed flow enhances inflammatory signaling and atherogenesis by increasing thioredoxin-1 level in endothelial cell nuclei.</a> PLOS ONE. 9 (9), e108346 (2014).</li><li>Kundumani-Sridharan, V., Dyukova, E., Hansen, D. E., Rao, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=12/15-Lipoxygenase+mediates+high-fat+diet-induced+endothelial+tight+junction+disruption+and+monocyte+transmigration:+a+new+role+for+15(S)-hydroxyeicosatetraenoic+acid+in+endothelial+cell+dysfunction.">12/15-Lipoxygenase mediates high-fat diet-induced endothelial tight junction disruption and monocyte transmigration: a new role for 15(S)-hydroxyeicosatetraenoic acid in endothelial cell dysfunction.</a> The Journal of Biological Chemistry. 288 (22), 15830-15842 (2013).</li><li>Liu, Z. H., 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=C1q/TNF-related+protein+1+promotes+endothelial+barrier+dysfunction+under+disturbed+flow.">C1q/TNF-related protein 1 promotes endothelial barrier dysfunction under disturbed flow.</a> Biochemical and Biophysical Research Communications. 490 (2), 580-586 (2017).</li><li>Wang, X. Q., 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=Thioredoxin+interacting+protein+promotes+endothelial+cell+inflammation+in+response+to+disturbed+flow+by+increasing+leukocyte+adhesion+and+repressing+Kruppel-like+factor+2.">Thioredoxin interacting protein promotes endothelial cell inflammation in response to disturbed flow by increasing leukocyte adhesion and repressing Kruppel-like factor 2.</a> Circulation Research. 110 (4), 560-568 (2012).</li><li>Mitra, S., Deshmukh, A., Sachdeva, R., Lu, J., Mehta, 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=Oxidized+low-density+lipoprotein+and+atherosclerosis+implications+in+antioxidant+therapy.">Oxidized low-density lipoprotein and atherosclerosis implications in antioxidant therapy.</a> The American Journal of the Medical Sciences. 342 (2), 135-142 (2011).</li><li>Stancel, N., 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=Interplay+between+CRP,+Atherogenic+LDL,+and+LOX-1+and+Its+Potential+Role+in+the+Pathogenesis+of+Atherosclerosis.">Interplay between CRP, Atherogenic LDL, and LOX-1 and Its Potential Role in the Pathogenesis of Atherosclerosis.</a> Clinical Chemistry. 62 (2), 320-327 (2016).</li><li>Nerem, R. M., Levesque, M. J., Cornhill, 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=Vascular+Endothelial+Morphology+as+an+Indicator+of+the+Pattern+of+Blood+Flow.">Vascular Endothelial Morphology as an Indicator of the Pattern of Blood Flow.</a> Journal of Biomechanical Engineering. 103 (3), 172-176 (1981).</li></ol>

The endothelium is exposed to numerous proatherogenic factors, including lipids, inflammatory mediators, and fluid shear stress1,11,12. Direct observation of endothelial cells in situ provides the special advantages to analyze changes in cell morphology, intercellular junctions, and molecule expression patterns in response to the injury stimuli.
Previous studies have provided two different en face imm...

The early changes in the vasculature primarily initiate in the endothelium, which functions as a selective barrier between the blood and the vessel wall with its intercellular tight junctional complexes1. Substantial evidence points to a critical role for the mechanical effects of blood flow in modulating endothelial function2. Fluid shear stress, a frictional force generated by blood flow, differentially shapes endothelial cell morphology and function, depending on the specific flow paradigms at different vascular sites2,3. Atherosclerotic lesions preferentially occur at sites of disturbed blood flow (d-flow), such as vessel curvatures, flow dividers, and branch points, as compared to regions of steady flow (s-flow), such as the straight segment of the artery. Therefore, direct observation of endothelial morphology and molecule expression patterns should provide important insights into the structural and functional phenotypes of endothelial cells under varying flow paradigms.
Cultured endothelial cells may not express the actual phenotype as they do in vivo partly due to the loss of impact of fluid shear stress, surrounding cytokines, and cell-cell or cell-extracellular matrix interactions. To aid this, the intact endothelial cell monolayer can be studied on transverse sections using classical immunohistochemistry. However, the endothelial monolayer is so thin and fragile that it usually cannot be observed clearly. En face immunohistochemistry has been used to observe the inner surface of the endothelium but is either complicated or erratic in its results because the endothelium is easily stripped from the underlying tissue, or just part of the arterial wall of rats or rabbits, whose walls are thick, is mounted4,5.
Mouse models have considerable advantages over other animals in many respects. Here, we employ a modified en face immunofluorescence technique to analyze endothelial cells of the aortic arch and thoracic aorta in C57BL/6 mouse. Such a technique has been widely used to study the endothelial pathophysiology in different flow patterns and in the development of atherosclerosis6,7,8,9,10. This method allows scientists to observe the entire surface of the endothelium clearly and to compare the expression patterns of a given protein in regions under different fluid shear stress.

<table border="0" cellpadding="0" cellspacing="0" style="width:655px;" width="655">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Antifade mountant</td>
			<td style="width:126px;">Servicebio</td>
			<td style="width:134px;">G1401</td>
			<td style="width:174px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Delicate Forceps</td>
			<td style="width:126px;">RWD Life Science</td>
			<td style="width:134px;">F11001-11</td>
			<td style="width:174px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Delicate Scissors</td>
			<td style="width:126px;">RWD Life Science</td>
			<td style="width:134px;">S12003-09</td>
			<td style="width:174px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Dissecting Forceps</td>
			<td style="width:126px;">RWD Life Science</td>
			<td style="width:134px;">F12005-10</td>
			<td style="width:174px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Mciro Spring Scissors</td>
			<td style="width:126px;">RWD Life Science</td>
			<td style="width:134px;">S11001-08</td>
			<td style="width:174px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Polyoxyethylene octyl phenyl ether (Triton X-100)</td>
			<td style="width:126px;">Amresco</td>
			<td style="width:134px;">M143</td>
			<td style="width:174px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Polysorbate 20 (Tween 20)</td>
			<td style="width:126px;">Amresco</td>
			<td style="width:134px;">0777</td>
			<td style="width:174px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">VCAM-1 antibody</td>
			<td style="width:126px;">Abcam</td>
			<td style="width:134px;">ab134047</td>
			<td style="width:174px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">VE-Cadherin antibody</td>
			<td style="width:126px;">BD Biosciences</td>
			<td style="width:134px;">555289</td>
			<td style="width:174px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Alexa Fluor 555 labeled anti-rabbit IgG</td>
			<td style="width:126px;">invitrogen</td>
			<td style="width:134px;">A-31572</td>
			<td style="width:174px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Alexa Fluor 488 labeled anti-rat IgG</td>
			<td style="width:126px;">invitrogen</td>
			<td style="width:134px;">A-21208</td>
			<td style="width:174px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Laser Scanning Microscope&#160;</td>
			<td>Carl Zeiss</td>
			<td style="width:134px;"></td>
			<td style="width:174px;"></td>
		</tr>
	</tbody>
</table>

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.

A 12-week-old C57BL/6 mouse was euthanized and perfused with normal saline containing 40 units/mL heparin and, then, prechilled 4% paraformaldehyde. The mouse aorta was exposed under a dissecting microscope (Figure 1), dissected, and cut open longitudinally (Figure 2). En face immunofluorescence staining of the vascular endothelial cells was performed as illustrated in Figure 3 and Table 1</s...

Watch this Scientific Journal Video about Using En Face Immunofluorescence Staining to Observe Vascular Endothelial Cells Directly at JoVE.com

Using En Face Immunofluorescence Staining to Observe Vascular Endothelial Cells Directly

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 ...

This protocol helps people analyze endothelial cell morphology, and the expression of molecules in regions, under different fluid shear stress. The main advantage of this technique is that it provides a direct assessment of vascular endothelial cells under different fluid shear stress. When performing this procedure, key steps are to use freshly prepared paraformaldehyde and to perform a good perfusion.Begin this procedure with anesthetization of a 12-week-old C57BL/6 mouse, as described in the text protocol. Confirm proper anesthetization by gently pinching the tail. Tape the mouse's paws to a stack of paper towels, with adhesive tape.Hold up the skin of the mouse with forceps, and cut the skin with a pair of scissors from the abdomen to the top of the thorax. Open the abdominal cavity, below the ribcage, with a sharp pair of scissors. Lift the sternum with forceps, and cut the diaphragm.Then cut away the ribcage to expose the thoracic cavity. Cut off the vena cava just above the liver with scissors. Pressure perfuse the arterial tree for five minutes, with pre-chilled normal saline containing 40 units per milliliter heparin, through the left ventricle, until the lungs and liver become pale.Then perfuse with pre-chilled 4%paraformaldehyde in PBS for four minutes. Remove all the muscles, organs, and fat, until the aorta is exposed. Place the mouse under a dissecting microscope.Use delicate forceps and a pair of delicate scissors to expose the aorta clearly under a dissecting microscope, and remove the connective tissue along the aorta, as cleanly as possible. Dissect the thoracic aorta from the heart to the celiac trunk with a pair of delicate scissors. Place the aorta into a six centimeter cell culture dish, with PBS.Cut open the aorta longitudinally, first along the lesser curve, and then use the microscissors to cut open the three branches of the aortic arch, including the innominate, left common carotid, and the left subclavian artery along the greater curve until the straight segment is met. Permeabilize the aorta with 1%polyoxyethylene octyl phenyl ether, in PBS for 10 minutes. Then block the aorta with 10%normal goat serum in tris-buffered saline containing 2.5%polysorbate 20 for one hour at room temperature, in a 12 well cell culture plate.Next incubate the aorta in the blocking buffer containing five grams per milliliter rabbit anti-VCAM1, and five grams per milliliter rat anti-VE-Cadherin, overnight at four degrees Celsius. After rinsing the sample three times with washing solution, apply the fluorescence conjugated secondary antibodies for one hour at room temperature, keeping in a dark place. Rinse another three times in the washing solution.Counterstain the aorta with DAPI for 10 minutes, keeping in a dark place. Then rinse the stained aorta three times in washing solution. Place the aorta on a cover slip with the lumenal surface downward, and move it slowly to antifade mounting solution that has been previously dropped on the cover slip.Gently stretch the aorta to keep the specimen flat. Invert the cover slip and put it on the glass slide. Take care to avoid any air bubbles between the specimen and the glass.Examine the aorta with a laser scanning confocal microscope. Analyze color intensities of different channels from the desired region in the en face images, with Image-Pro Plus software. En face immunofluorescence staining images of vascular endothelial cells from the mouse aorta are shown.En face immunofluorescence of the vascular cell adhesion protein one, and expression with VE-Cadherin as an endothelial marker, are shown under varying flow patterns from different regions of the mouse aorta. DAPI was also counterstained to show the cell nuclei for better visualization. When looking through the Z stacks under the microscope, the endothelial and smooth muscle cells can be easily distinguished by their cell nuclei morphology.The endothelial cell nuclei are oval shaped, and bigger than the spindle shaped, smooth muscle cell nuclei. Expression of VCAM-1 was more abundant in regions under disturbed flow, at lesser curvature of the aorta arch, compared with regions under steady flow, at greater curvature of the aorta arch, and at the thoracic aorta. Here areas of lesser curvature of a mouse aorta are indicated as d-flow areas, and the greater curvature and thoracic.With the right modification, this technique can be also applied to analysis of vascular permeability, and the position of macromolecules in the endothelial cell intima.

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14.0K vistas.  Shanghai Jiao Tong University School of Medicine. Este protocolo ayuda a las personas a analizar la morfología celular endotelial, y la expresión de moléculas en regiones, bajo diferentes estrés de cizallamiento de fluidos. La principal ventaja de esta técnica es que proporciona una evaluación directa de las células endoteliales vasculares bajo diferentes tensiones de cizallamiento de fluidos. Al realizar este procedimiento, los pasos clave son utilizar paraformaldehído recién preparado y realizar una buena perfusión.Comienc...