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.</li>
	<li>Tape the mouse&#39;s paws to a stack of paper towels with adhesive tapes.</li>
	<li>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.</li>
	<li>Open the abdominal cavity below the ribcage with a sharp pair of scissors.</li>
	<li>Lift the sternum with forceps and cut the diaphragm; then, cut away the ribcage to expose the thoracic cavity.</li>
	<li>Cut off the vena cava just above the liver, with scissors.</li>
	<li>Pressure perfuse (100 mmHg) the arterial tree for 5 min with prechilled normal saline containing 40 units/mL heparin through the left ventricle until the lungs and liver become pale. Then, perfuse with prechilled 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4) for 3 min.</li>
	<li>Remove all the muscles, organs, and fat until the aorta is exposed.</li>
	<li>Place the mouse under a dissecting microscope.</li>
</ol>
2. Dissection and longitudinally opening of the aorta
<ol>
	<li>Expose the aorta clearly under a dissecting microscope and remove the connective tissues along the aorta as clean as possible, with delicate forceps and a pair of delicate scissors (Figure 1).</li>
	<li>Dissect the thoracic aorta from the heart to the celiac trunk with a pair of delicate scissors and put the aorta into a 6 cm cell culture dish with PBS. Cut open the aorta longitudinally, along the lesser curve, and along the greater curve until the straight segment is met. Cut open the three branches of the aortic arch, including the innominate, left common carotid and the left subclavian artery, with microscissors, as shown in Figure 2.</li>
</ol>
3. Pretreatment and immunostaining of the aorta
<ol>
	<li>Permeabilize the aorta with 0.1% polyoxyethylene octyl phenyl ether in PBS for 10 min and block it with 10% normal goat serum in Tris-buffered saline (TBS) containing 2.5% polysorbate 20 for 1 h at room temperature in a 12-well cell culture plate.</li>
	<li>Next, incubate the aorta with 5 g/mL rabbit anti-VCAM-1 and 5 g/mL rat anti-VE-cadherin in the blocking buffer, overnight at 4 &#176;C.</li>
	<li>After rinsing the sample 3x with washing solution (TBS containing 2.5% polysorbate 20), apply the fluorescence-conjugated secondary antibodies (1:1,000 dilution, Alexa Fluor 555-labeled anti-rabbit IgG and Alexa Fluor 488-labeled anti-rat IgG) for 1 h at room temperature. Rinse 3x in the washing solution.</li>
	<li>Counterstain the aorta with 4&#39;,6-diamidino-2-phenylindole (DAPI) (1 &#181;g/mL) for 10 min and rinse it 3x in the washing solution.</li>
</ol>
4. Mounting of the aorta on the glass slide
<ol>
	<li>Place the aorta on a coverslip with the luminal surface downward and move it slowly to the antifade mounting solution previously dropped on the coverslip. Gently stretch the aorta to keep the specimen flat.</li>
	<li>Inverse the coverslip and put it on the slide glass. Care must be taken not to allow any air bubbles to remain between the specimen and the glass.</li>
</ol>
5. Observation of the aorta
<ol>
	<li>Examine the aorta with a laser-scanning confocal microscope.</li>
	<li>Analyze color intensities of different channels from the desired region in the en face images with Image-Pro Plus software.</li>
</ol>

<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. 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The authors have nothing to disclose.

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 immunohistochemical techniques to observe the endothelium of the arterial wall4,5. One is to obtain an endothelial monolayer by stripping the endothelium from the vessel wall by a special technique, which is difficult to master and could result in the loss of much endothelium at the branching sites of the artery4. The other is mounting the whole arterial wall as we described. Whole-mount preparation is easy to perform and can maintain the whole endothelial monolayer in rabbit and rats5,13. However, spreading the whole mouse aortic specimen flat is not easy, and failure to do so may largely affect the quality of the observation. The preparation of the aortic specimen as described here, with the curvatures cut open and the luminal surface faced downward on the coverslip, makes it easier to spread the aortic specimen flat on the slide glass. Moreover, a laser-scanning confocal microscope is required to better focus on the thin endothelial cell monolayer lining the blood vessel.
A key point of this technique is to stretch the aortic specimen as flat as possible to get a better observation of the aorta. In addition, heparin should be added to normal saline before perfusion, and the aortic specimen should be treated gently throughout the process. Paraformaldehyde should be freshly prepared and kept at 4 &#176;C until usage.
There are limitations to this technique. First, the capacity of en face immunofluorescence to detect subendothelial substance, such as deposited low-density lipoprotein, is limited when compared to regular immunohistochemistry of tissue sections. Second, the fluorescent signals are readily bleached after a scan by the confocal microscope, especially when performing z-axis analysis. Moreover, the expression of the target molecule can only be semiquantified, according to its fluorescent intensity.
In summary, this modified&#160;en face immunofluorescence staining technique provides an easy way to analyze the morphology and protein expression pattern of vascular endothelial cells in regions under different blood flow paradigms6,7 and in the pathogenesis of atherosclerosis.

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><tbody><tr><td>Antifade mountant</td><td>Servicebio</td><td>G1401</td><td></td></tr><tr><td>Delicate Forceps</td><td>RWD Life Science</td><td>F11001-11</td><td></td></tr><tr><td>Delicate Scissors</td><td>RWD Life Science</td><td>S12003-09</td><td></td></tr><tr><td>Dissecting Forceps</td><td>RWD Life Science</td><td>F12005-10</td><td></td></tr><tr><td>Mciro Spring Scissors</td><td>RWD Life Science</td><td>S11001-08</td><td></td></tr><tr><td>Polyoxyethylene octyl phenyl ether (Triton X-100)</td><td>Amresco</td><td>M143</td><td></td></tr><tr><td>Polysorbate 20 (Tween 20)</td><td>Amresco</td><td>0777</td><td></td></tr><tr><td>VCAM-1 antibody</td><td>Abcam</td><td>ab134047</td><td></td></tr><tr><td>VE-Cadherin antibody</td><td>BD Biosciences</td><td>555289</td><td></td></tr><tr><td>Alexa Fluor 555 labeled anti-rabbit IgG</td><td>invitrogen</td><td>A-31572</td><td></td></tr><tr><td>Alexa Fluor 488 labeled anti-rat IgG</td><td>invitrogen</td><td>A-21208</td><td></td></tr><tr><td>Laser Scanning Microscope&amp;nbsp;</td><td>Carl Zeiss</td><td></td><td></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. En face immunofluorescence of the vascular cell adhesion protein-1 (VCAM-1) expression with VE-cadherin as endothelial marker was shown under varying flow patterns from different regions of the mouse aorta. DAPI was also counterstained to show the cell nuclei for better visualization. The endothelial and smooth muscle cells can be easily distinguished from the morphology of the cell nuclei when looking through the z-stacks under the microscope since the endothelial cell nuclei are oval shaped and bigger than the spindle-shaped smooth muscle cell nuclei. The representative en face&#160;images are shown in Figure 4. The aorta was examined by the LSM 710 Laser Scanning Microscope (Table of Materials) with a FLUAR 40x/1,3 oil lens. From the en face immunofluorescence staining, we can clearly and directly observe that the expression of VCAM-1 was more abundant in regions under disturbed flow (lesser curvature of the aorta arch) than in those under steady flow (greater curvature of the aorta arch and the thoracic aorta).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59325/59325fig1.jpg" /> 
Figure 1: Exposing the mouse aorta under a dissecting microscope. Remove the connective tissues along the aorta as cleanly as possible. a = innominate artery; b = left common carotid artery; c = left subclavian artery. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59325/59325fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59325/59325fig2.jpg" /> 
Figure 2: Dissection of the mouse thoracic aorta and cutting it open longitudinally. (A) The thoracic aorta from the heart to the celiac trunk was cut open along the lesser curve longitudinally, and along the greater curve, until the straight segment was met. The three branches of the aortic arch, including the innominate, left common carotid, and left subclavian artery, were also cut open with microscissors. Red dashed lines indicate the cutting line. (B)&#160;The aorta was opened and spread flat on the glass slides. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59325/59325fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59325/59325fig3.jpg" /> 
Figure 3: Schema for en face&#160;immunofluorescence staining of vascular endothelial cells. First, permeabilize the dissected aorta with 0.1% polyoxyethylene octyl phenyl ether in PBS for 10 min and block it with 10% normal goat serum in TBS containing 2.5% polysorbate 20 for 1 h at room temperature in a 12-well cell culture plate. Next, incubate the aorta with primary antibody in the blocking buffer overnight at 4 &#176;C, and rinse it three times with washing solution (TBS containing 2.5% polysorbate 20). Then, apply fluorescence-conjugated secondary antibodies for 1 h at room temperature and rinse three times. Counterstain the aorta with DAPI for 10 min and rinse it three times in the washing solution. At last, mount the aorta on a glass slide and observe the aorta by a laser-scanning confocal microscope. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59325/59325fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59325/59325fig4.jpg" /> 
Figure 4: Representative en face&#160;staining results. (A) En face&#160;immunofluorescence analysis of the lesser curvature of a mouse aorta (d-flow areas) and the greater curvature or thoracic aorta (s-flow areas) to compare the endothelial VCAM-1 expression under different flow paradigms. Endothelial cell morphology is shown by VE-cadherin staining. (B) Representative d-flow areas (lesser curvature) and s-flow areas (greater curvature and thoracic aorta) are indicated by arrows. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59325/59325fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Step</td>
			<td>Process</td>
			<td>Buffer</td>
			<td>Temperature</td>
			<td>Time</td>
		</tr>
		<tr>
			<td>1</td>
			<td>Permeabilize</td>
			<td>0.1%&#160; polyoxyethylene octyl phenyl ether in PBS&#160;</td>
			<td>Room temperature</td>
			<td>10 min</td>
		</tr>
		<tr>
			<td>2</td>
			<td>Blocking</td>
			<td>10% normal goat serum in Tris-buffered saline (TBS) containing 2.5% polysorbate 20</td>
			<td>Room temperature</td>
			<td>1 h</td>
		</tr>
		<tr>
			<td>3</td>
			<td>First antibody (5 g/mL)</td>
			<td>Blocking buffer</td>
			<td>4 &#176;C</td>
			<td>Overnight</td>
		</tr>
		<tr>
			<td>4</td>
			<td>Rinse</td>
			<td>TBS containing 2.5% polysorbate 20</td>
			<td>Room temperature</td>
			<td>5 min, 3 times</td>
		</tr>
		<tr>
			<td>5</td>
			<td>Fluorescence secondary antibody (1:1000)</td>
			<td>Blocking buffer</td>
			<td>Room temperature</td>
			<td>1 h</td>
		</tr>
		<tr>
			<td>6</td>
			<td>Rinse</td>
			<td>TBS containing 2.5% polysorbate 20</td>
			<td>Room temperature</td>
			<td>5 min, 3 times</td>
		</tr>
	</tbody>
</table>
Table 1: Detailed information about the en faceimmunofluorescence staining procedure.

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 ...

using-en-face-immunofluorescence-staining-to-observe-vascular

Universidad San Sebastian

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JoVE Journal

Biology

14.9K Views.  Shanghai Jiao Tong University School of Medicine. 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.

Video: Using En Face Immunofluorescence Staining to Observe Vascular Endothelial Cells Directly

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

Immunology and Infection

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to extravasate and form a secondary niche.&#160;The extravasation of tumor cells from the blood vascular system can be studied using endothelial cells (ECs) and tumor cells obtained from different cell lines.&#160;Initial studies were conducted using static conditions but it has been well documented that ECs behave differently under physiological flow conditions.&#160;Therefore, different flow chamber assemblies are currently being used to studying cancer cell interactions with ECs.&#160;Current flow chamber assemblies offer reproducible results using either different cell lines or fluid at different shear stress conditions.&#160;However, to observe and study interactions with rare cells such as circulating tumor cells (CTCs), certain changes are required to be made to the conventional flow chamber assembly.&#160;CTCs are a rare cell population among millions of blood cells. Consequently, it is difficult to obtain a pure population of CTCs.&#160;Contamination of CTCs with different types of cells normally found in the circulation is inevitable using present enrichment or depletion techniques.&#160;In the present report, we describe a unique method to fluorescently label circulating prostate cancer cells and study their interactions with ECs in a self-assembled flow chamber system.&#160;This technique can be further applied to observe interactions between prostate CTCs and any protein of interest.

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Our report describes a unique method to visualize and analyze CTC/EC interactions in prostate cancer under physiological flow conditions.

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to ...

Medicine

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial cells regulate permeability of the endothelium, and many studies have indicated the important contribution of the actin cytoskeleton to determining junctional integrity1-5. A cortical actin belt is thought to be important for the maintenance of stable junctions1, 2, 4, 5. In contrast, actin stress fibers are thought to generate centripetal tension within endothelial cells that weakens junctions2-5. Much of this theory has been based on studies in which endothelial cells are treated with inflammatory mediators known to increase endothelial permeability, and then fixing the cells and labeling F-actin for microscopic observation. However, these studies provide a very limited understanding of the role of the actin cytoskeleton because images of fixed cells provide only snapshots in time with no information about the dynamics of actin structures5. 
Live-cell imaging allows incorporation of the dynamic nature of the actin cytoskeleton into the studies of the mechanisms determining endothelial barrier integrity. A major advantage of this method is that the impact of various inflammatory stimuli on actin structures in endothelial cells can be assessed in the same set of living cells before and after treatment, removing potential bias that may occur when observing fixed specimens. Human umbilical vein endothelial cells (HUVEC) are transfected with a GFP-&beta;-actin plasmid and grown to confluence on glass coverslips. Time-lapse images of GFP-actin in confluent HUVEC are captured before and after the addition of inflammatory mediators that elicit time-dependent changes in endothelial barrier integrity. These studies enable visual observation of the fluid sequence of changes in the actin cytoskeleton that contribute to endothelial barrier disruption and restoration. 
Our results consistently show local, actin-rich lamellipodia formation and turnover in endothelial cells. The formation and movement of actin stress fibers can also be observed. An analysis of the frequency of formation and turnover of the local lamellipodia, before and after treatment with inflammatory stimuli can be documented by kymograph analyses. These studies provide important information on the dynamic nature of the actin cytoskeleton in endothelial cells that can used to discover previously unidentified molecular mechanisms important for the maintenance of endothelial barrier integrity.

Microscopic imaging of live endothelial cells expressing GFP-actin allows characterization of dynamic changes in cytoskeletal structures. Unlike techniques that use fixed specimens, this method provides a detailed assessment of temporal changes in the actin cytoskeleton in the same cells before, during, and after various physical, pharmacological, or inflammatory stimuli.

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell ...

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.

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.

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

Isolation and Magnetic Separation of Murine CD34+ Stem Cells from the Vascular Wall

Immunomagnetic Isolation of the Vascular Wall-Resident CD34+ Stem Cells from Mice

Resident CD34+ vascular wall-resident stem and progenitor cells (VW-SCs) are increasingly recognized for their crucial role in regulating vascular injury and repair. Establishing a stable and efficient method to culture functional murine CD34+ VW-SCs is essential for further investigating the mechanisms involved in the proliferation, migration, and differentiation of these cells under various physiological and pathological conditions. The described method combines magnetic bead screening and flow cytometry to purify primary cultured resident CD34+ VW-SCs. The purified cells are then functionally identified through immunofluorescence staining and Ca2+ imaging. Briefly, vascular cells from the adventitia of the murine aorta and mesenteric artery are obtained through tissue block attachment, followed by subculturing until reaching a cell count of at least 1 &#215; 107. Subsequently, CD34+ VW-SCs are purified using magnetic bead sorting and flow cytometry. Identification of CD34+ VW-SCs involves cellular immunofluorescence staining, while functional multipotency is determined by exposing cells to a specific culture medium for oriented differentiation. Moreover, functional internal Ca2+ release and external Ca2+ entry is assessed using a commercially available imaging workstation in Fura-2/AM-loaded cells exposed to ATP, caffeine, or thapsigargin (TG). This method offers a stable and efficient technique for isolating, culturing, and identifying vascular wall-resident CD34+ stem cells, providing an opportunity for in vitro studies on the regulatory mechanisms of VW-SCs and the screening of targeted drugs.

Author Spotlight: Enhanced Isolation and Characterization of Vascular Wall Resident Murine CD34+ Stem Cells Using Magnetic Bead Screening-Coupled Flow Cytometry

This study has established a stable and efficient method for the isolation, culture, and functional determination of vascular wall-resident CD34+ stem cells (CD34+ VW-SCs). This easy-to-follow and time-effective isolation method can be utilized by other investigators to study the potential mechanisms involved in cardiovascular diseases.

Resident CD34+ vascular wall-resident stem and progenitor cells (VW-SCs) are increasingly recognized for their crucial role in regulating vascular injury ...

Cytoskeleton and Focal Adhesion Organization Assay: An Immunofluorescence-based Method to Study Cell Adhesion and Spreading on Substrates

Source: Ali, M. Y et al., <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/52532"> Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry.</a> J. Vis. Exp. (2015)
This video describes the technique of studying the cytoskeleton and focal adhesions in primary human colon cancer cells to understand its variations depending on substrate rigidity. This culture of cancer cells on soft and hard substrates allows various downstream biophysical measurements for cancer prognosis.

Source: Ali, M. Y et al.,  Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for ...

JoVE Encyclopedia of Experiments

Cancer Research

Colorectal Cancer

Assays & techniques

Immunofluorescence Staining of Gel-Embedded Patient-Derived Diseased Fibrovascular Tissue

<a href='https://app-jove-com.bdigitaluss.remotexs.co/v/59090/an-ex-vivo-tissue-culture-model-for-fibrovascular-complications'>Source: Gucciardo, E., et. al., An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy. J. Vis. Exp. (2019)</a>This video details an immunostaining protocol for ex vivo cultures of fibrovascular tissue embedded in fibrin gel. This enables detailed visualization of vascular changes associated with proliferative diabetic retinopathy

Source: Gucciardo, E., et. al., An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy. J. Vis. Exp. ...

Using En Face Immunofluorescence Staining to Observe Vascular Endothelial Cells Directly

Summary

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Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

En Face Preparation of Mouse Blood Vessels

Immunomagnetic Isolation of the Vascular Wall-Resident CD34⁺ Stem Cells from Mice

Cytoskeleton and Focal Adhesion Organization Assay: An Immunofluorescence-based Method to Study Cell Adhesion and Spreading on Substrates

Immunofluorescence Staining of Gel-Embedded Patient-Derived Diseased Fibrovascular Tissue