Using En Face Immunofluorescence Staining to Observe Vascular Endothelial Cells Directly

Chang Li; Zhu Hui Liu; Jia Wei Chen; Xin Yi Shu; Ying Shen; Feng Hua Ding; Rui Yan Zhang; Wei Feng Shen; Lin Lu; Xiao Qun Wang

doi:10.3791/59325

Using En Face Immunofluorescence Staining to Observe Vascular Endothelial Cells Directly

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Biology

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JoVE Journal Biology

Using En Face Immunofluorescence Staining to Observe Vascular Endothelial Cells Directly

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06:09 min

August 20, 2019

DOI: 10.3791/59325-v

Chang Li*^1,2, Zhu Hui Liu*^1,2, Jia Wei Chen^1,2, Xin Yi Shu^1,2, Ying Shen¹, Feng Hua Ding¹, Rui Yan Zhang¹, Wei Feng Shen¹, Lin Lu¹, Xiao Qun Wang¹

¹Department of Cardiology, Ruijin Hospital,Shanghai Jiao Tong University School of Medicine, ²Institute of Cardiovascular Diseases,Shanghai Jiao Tong University

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

What is the significance of the endothelial cells in the aorta?

Endothelial cells play a critical role in maintaining vascular health and are involved in the pathophysiology of atherosclerosis.

How does fluid shear stress affect endothelial cells?

Fluid shear stress influences endothelial cell morphology and function, impacting their response to inflammation and atherosclerosis.

What advantages does immunofluorescence staining offer?

Immunofluorescence staining provides a highly detailed visualization of cell surface markers and allows for direct assessment of cellular conditions.

How does this method help in atherosclerosis research?

It enables researchers to study the effects of different hemodynamic environments on endothelial cell behavior and phenotypic changes linked to atherosclerosis.

What are the key steps in the staining protocol?

Key steps include proper perfusion, blocking, and staining with fluorescently labeled antibodies, followed by imaging under a confocal microscope.

Can this technique be applied to other vascular studies?

Yes, with appropriate modifications, it can be adapted for studying vascular permeability and interactions with macromolecules.

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.

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En Face Immunofluorescence Endothelial Cell Morphology Fluid Shear Stress Vascular Endothelial Cells Paraformaldehyde C57BL/6 Mouse Pressure Perfusion Thoracic Aorta Dissecting Microscope Aortic Arch Rabbit Anti-VCAM1 Blocking Buffer PBS Normal Goat Serum