Using En Face Immunofluorescence Staining to Observe Vascular Endothelial Cells Directly

Summary

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.

Abstract

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.

Introduction

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 sites^2,3. 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 mounted^4,5.

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 atherosclerosis^6,7,8,9,10. 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.

Protocol

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

1. 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.
2. Tape the mouse's paws to a stack of paper towels with adhesive tapes.
3. 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.
4. Open the abdominal cavity below the ribcage with a sharp pair of scissors.
5. Lift the sternum with forceps and cut the diaphragm; then, cut away the ribcage to expose the thoracic cavity.
6. Cut off the vena cava just above the liver, with scissors.
7. 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.
8. Remove all the muscles, organs, and fat until the aorta is exposed.
9. Place the mouse under a dissecting microscope.

2. Dissection and longitudinally opening of the aorta

1. 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).
2. 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.

3. Pretreatment and immunostaining of the aorta

1. 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.
2. 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 °C.
3. 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.
4. Counterstain the aorta with 4',6-diamidino-2-phenylindole (DAPI) (1 µg/mL) for 10 min and rinse it 3x in the washing solution.

4. Mounting of the aorta on the glass slide

1. 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.
2. 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.

5. Observation of the aorta

1. Examine the aorta with a laser-scanning confocal microscope.
2. Analyze color intensities of different channels from the desired region in the en face images with Image-Pro Plus software.

Results

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

Discussion

The endothelium is exposed to numerous proatherogenic factors, including lipids, inflammatory mediators, and fluid shear stress^1,11,12. 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...

Materials

Name	Company	Catalog Number	Comments
Antifade mountant	Servicebio	G1401
Delicate Forceps	RWD Life Science	F11001-11
Delicate Scissors	RWD Life Science	S12003-09
Dissecting Forceps	RWD Life Science	F12005-10
Mciro Spring Scissors	RWD Life Science	S11001-08
Polyoxyethylene octyl phenyl ether (Triton X-100)	Amresco	M143
Polysorbate 20 (Tween 20)	Amresco	0777
VCAM-1 antibody	Abcam	ab134047
VE-Cadherin antibody	BD Biosciences	555289
Alexa Fluor 555 labeled anti-rabbit IgG	invitrogen	A-31572
Alexa Fluor 488 labeled anti-rat IgG	invitrogen	A-21208
Laser Scanning Microscope	Carl Zeiss