Using En Face Immunofluorescence Staining to Observe Vascular Endothelial Cells Directly
August 20th, 2019
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.
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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Overview
This study presents a protocol for immunofluorescence staining to directly observe endothelial cells of the mouse aorta. The technique is effective for analyzing the cellular and molecular phenotype of these cells under different fluid shear stress conditions, particularly in relation to atherosclerosis development.
Key Study Components
Research Area
- Endothelial cell biology
- Atherosclerosis research
- Immunofluorescence techniques
Background
- Importance of endothelial cells in vascular biology
- Role of shear stress in endothelial function
- Connection between flow patterns and atherosclerosis development
Methods Used
- Immunofluorescence staining protocol
- Mouse model (C57BL/6)
- Confocal microscopy for imaging
Main Results
- Direct assessment of endothelial cells using fluorescent markers
- Increased VCAM-1 expression in disturbed flow areas
- Clear distinction between endothelial and smooth muscle cell nuclei morphology
Conclusions
- This study provides a reliable method for examining endothelial cell responses to hemodynamic forces.
- Findings are significant for understanding vascular biology and the mechanisms underlying atherosclerosis.
Frequently Asked Questions
Executive Industry Relevance
Direct visualization of intact endothelial monolayers under physiological shear stress enables mechanistic de-risking of vascular targets in atherosclerosis research. This approach supports predictive confidence in target validation by linking hemodynamic forces to molecular phenotypes in situ. It provides a disease-relevant system for early-stage pathophysiological insight without requiring genetic manipulation or pharmacological perturbation.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by correlating disturbed flow patterns with VCAM-1 upregulation as an early inflammatory marker.
- Operational Value: Provides a reliable, reproducible method for functional target validation using endogenous endothelial markers like VE-Cadherin.
- Predictive Value: Supports portfolio triage by identifying molecular responses that precede lesion formation in preclinical atherosclerosis models.
Screening & Assay Development
- Assay Readiness: Generates standardized, quantitative immunofluorescence outputs suitable for high-content analysis of endothelial phenotypes.
- Scalability: Allows parallel assessment of multiple aortic regions under defined flow conditions, enabling comparative screening.
- Assay Standardization: Uses consistent perfusion, blocking, and staining protocols to minimize variability across experiments and laboratories.
Translational & Preclinical Research
- Disease Relevance: Directly models early atherosclerotic susceptibility by examining endothelial responses in regions prone to plaque initiation.
- Translational Continuity: Bridges hemodynamic mechanobiology with molecular readouts relevant to human vascular pathology.
- Risk-Adjusted Advancement: Enables mechanistic de-risking of targets by validating their regulation under pathophysiological shear stress before in vivo efficacy testing.
Pipeline & Workflow Integration
The technique fits within the discovery continuum from target hypothesis testing through phenotypic screening to preclinical validation, particularly for vasculature-focused programs.
- Discovery Biology: Supports hypothesis testing by enabling direct observation of endothelial activation states under controlled shear stress.
- Screening: Delivers quantitative, multiplexable readouts (e.g., VCAM-1, VE-Cadherin, nuclear morphology) for comparing compound or genetic effects on endothelial phenotype.
- Analytics: Provides spatially resolved, intensity-based measurements that allow statistical comparison of molecular expression across aortic regions.
- Translational Research: Connects in vitro flow models to in vivo-like endothelial responses, enhancing predictive value for preclinical studies.
- Enterprise Reuse: Establishes a reusable vascular phenotyping platform applicable across multiple atherosclerosis, hypertension, or vascular inflammation projects.
Operational & Enterprise Impact
- Scientific Value: Reduces mechanistic ambiguity by linking hemodynamic forces to specific endothelial molecular phenotypes in intact tissue.
- Operational Value: Ensures standardization and reproducibility through defined perfusion, fixation, and staining steps.
- Strategic Value: Improves go/no-go decisions by validating target engagement under pathophysiologically relevant mechanical cues.
- Portfolio Impact: Enables risk-adjusted prioritization of targets based on their regulation in early atherosclerotic endothelial phenotypes.
Implementation Considerations
- Requires expertise in mouse vascular dissection, perfusion techniques, and confocal microscopy.
- Dependent on access to perfusion equipment, dissecting microscopes, and laser scanning confocal systems with spectral unmixing.
- Necessitates standardization of perfusion pressure, fixative temperature, and antibody titration across users and sites.
- Adaptation to other vascular beds (e.g., carotid, coronary) may require optimization of dissection and permeabilization steps.
- Limited to endpoint analysis; does not support real-time dynamic imaging of endothelial responses under flow.
