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. 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 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 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><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</s...

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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 ...

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14.8K 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

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Bioengineering

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological synapses&#39;. Within these intimate cell-cell interfaces discrete sub-cellular clusters of MHC/Ag-TCR, F-actin, adhesion and signaling molecules form and remodel rapidly. These dynamics are thought to be critical determinants of both the efficiency and quality of the immune responses that develop and therefore of protective versus pathologic immunity. Current understanding of immunological synapses with physiologic APCs is limited by the inadequacy of the obtainable imaging resolution. Though artificial substrate models (e.g., planar lipid bilayers) offer excellent resolution and have been extremely valuable tools, they are inherently non-physiologic and oversimplified. Vascular and lymphatic endothelial cells have emerged as an important peripheral tissue (or stromal) compartment of &#39;semi-professional APCs&#39;. These APCs (which express most of the molecular machinery of professional APCs) have the unique feature of forming virtually planar cell surface and are readily transfectable (e.g., with fluorescent protein reporters). Herein a basic approach to implement endothelial cells as a novel and physiologic &#39;planar cellular APC model&#39; for improved imaging and interrogation of fundamental antigenic signaling processes will be described.

Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological ...

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 ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

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 ...

Assessing Immunological Synapse Topology through Live-Cell Imaging

Source: Martinelli, R. et al., <a href="https://app.jove.com/v/53288">An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics.</a>&#160;J. Vis. Exp. (2015)
This video demonstrates live-cell imaging of immunological synapse topology, investigating the interactions between T lymphocytes and epithelial cells carrying fluorescent antigens. Fluorescence microscopy reveals red cytoplasm displacement and yellow membrane rings in endothelial cells, indicating the formation of immunological synapses.

1. Preparing Human CD4+&#160;Th1 Effector/Memory T Cells

	Apply a tourniquet to the arm of the donor, wipe the vein with alcohol, and insert a needle. ...

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Immunology

Immunodiagnostics

Imaging and Visualization Techniques

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

<a href='https://app.jove.com/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

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Chapters in this video

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

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

Micropatterning and Assembly of 3D Microvessels

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

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

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

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

En Face Preparation of Mouse Blood Vessels

Assessing Immunological Synapse Topology through Live-Cell Imaging

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