Visualization and Analysis of Pharyngeal Arch Arteries using Whole-mount Immunohistochemistry and 3D Reconstruction

Summary

Here, we describe a protocol to visualize and analyze the pharyngeal arch arteries 3, 4, and 6 of mouse embryos using whole-mount immunofluorescence, tissue clearing, confocal microscopy, and 3D reconstruction.

Abstract

Improper formation or remodeling of the pharyngeal arch arteries (PAAs) 3, 4, and 6 contribute to some of the most severe forms of congenital heart disease. To study the formation of PAAs, we developed a protocol using whole-mount immunofluorescence coupled with benzyl alcohol/benzyl benzoate (BABB) tissue clearing, and confocal microscopy. This allows for the visualization of the pharyngeal arch endothelium at a fine cellular resolution as well as the 3D connectivity of the vasculature. Using software, we have established a protocol to quantify the number of endothelial cells (ECs) in PAAs, as well as the number of ECs within the vascular plexus surrounding the PAAs within pharyngeal arches 3, 4, and 6. When applied to the whole embryo, this methodology provides a comprehensive visualization and quantitative analysis of embryonic vasculature.

Introduction

During mouse embryogenesis, pharyngeal arch arteries (PAAs) arise as symmetrical, bi-lateral pairs of arteries that connect the heart with the dorsal aortae¹. As the embryo develops, the first and second pairs of PAAs regress, while the 3^rd, 4^th, and 6^th PAAs undergo a series of asymmetrical remodeling events to form the aortic arch arteries².

The PAAs 3, 4 and 6 develop via vasculogenesis, which is the de novo formation of blood vessels³. Defects in the formation or remodeling of these arch arteries give rise to various congenital heart....

Protocol

Animal use and procedures were approved by the Institutional Animal Care and Use Committee at Rutgers University.

1. Preparation of solutions

1. Prepare 1 L of phosphate buffered saline with 0.1% Triton-X-100 (PBST) and filter sterilize. This solution can be stored at room temperature (RT) for at least a year.
2. Prepare 600 µL of blocking buffer consisting of 10% of normal donkey serum in PBST. Make this solution fresh each time.
3. Prepare 50 mL of the following m.......

Discussion

The ability to visualize the endothelium in mouse embryos in 3D has provided new insights into their development³. Here we present a protocol that allows for high-resolution 3D imaging of embryos, visualization of vascular connectivity, and quantitative analyses of PAA formation. This protocol can be employed to see how genetic alterations or environmental insults impact PAA development. The procedure reported here uses antibodies against VEGFR2 and ERG to visualize PAA formation and quantify EC n.......

Materials

Name	Company	Catalog Number	Comments
10x PBS	MP Biomedicals	PBS10X02
20x water immersion objective	Nikon	MRD77200
Agarose	Bio-Rad Laboratories	1613101
Alexa Fluor 488 anti-goat	Invitrogen	A-11055
Alexa Fluor 555 anti-mouse	Invitrogen	A-31570
Analysis Software	Imaris 9.2.0
Benzyl Alcohol	Sigma-Aldrich	305197
Benzyl Benzoate	Sigma-Aldrich	8.18701.0100
Cover Slips	VWR	16004-312
DAPI (5 mg/mL stock)	Fisher Scientific	D3571
Eppendorf Tubes (2.0 mL)	Fisher Scientific	05-408-138
Ethanol	VWR	89370-084
Falcon tubes (50 mL)	Corning	352098
Fast wells	Grace Bio Labs	664113
Forceps	Roboz	RS-5015
Goat anti-VEGFR2	R&D Systems, Inc.	AF644
Methanol	VWR	BDH1135-4LP
Microscope	Nikon	A1HD25
Mouse anti-ERG	Abcam	ab214341
Normal Donkey Serum	Sigma-Aldrich	D9663
Paraformaldehyde	Electron Microscopy Sciences	15710
Pasteur pipets	Fisher Scientific	13-678-20D
Petri dishes (35 mm)	Genesee Scientific	32-103
Petri dishes (60 mm)	Genesee Scientific	32-105
Plastic Molds	VWR	18000-128
Scapels	Exelint International Co.	29552
Triton-X-100	Fisher Scientific	BP 151-500