Injection of Microbubbles into Isolated Mouse Embryos: A Technique to Deliver Microbubble Contrast Agents into the Vasculature of Living Murine Embryos

Abstract

Source: Denbeigh, J. M. et al. Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound. J. Vis. Exp. (2015)

This video demonstrates a procedure to inject microbubble contrast agents into an isolated late gestation stage mouse embryo. The microbubble contrast agent-based ultrasound imaging helps visualize and characterize the vascular environment of the embryo.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Microbubble Preparation

1. Preparing microbubbles for injection
   NOTE: The assistant performs this step when ‘Injection of Microbubbles (MB) into Embryos’ is commenced. Each embryo is injected once, with the type of microbubble chosen for each injection to be randomly selected by the assistant such that all types of microbubbles are evenly distributed throughout the procedure but given in a random order. Ensure the surgeon is blind to ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ice
0.9% sodium chloride (saline)	Hospira	0409-7984-11
Phosphate Buffered Saline [1x]	Sigma	D8537	1x, w/o calcium chloride & magnesium chloride
Ultrasound contrast agent, target ready and untargeted	MicroMarker; VisualSonics Inc.
Ultrasound gel (Aquasonic 100, colourless)	CSP Medical	133-1009
Ice bucket	Cole Parmer	RK 06274-01
Imaging Platform	VisualSonics Inc.	Integrated Rail System
Light source, fiber-optic	Fisher Scientific	12-562-36	Ideally has adjustable arms
Luers (12), polypropylene barbed female ¼-28 UNF thread	Cole Parmer	45500-30
Micro-ultrasound system, high-frequency	VisualSonics Inc.	Vevo2100
Needles, 21 gauge  (1”)	VWR	305165
Perforated spoon (Moria)	Fine Science Tools	MC 17 10373-17
Pins (6), black anodized minutien 0.15 mm	Fine Science Tools	26002-15
Syringes, 1 mL Normject	Fisher	14-817-25
Syringe infusion pump	Bio-lynx	NE-1000
Pipettors [2-20 μL, 20-200 μL, 100-1,000 μL]	Eppendorf	Research Plus adjustable 3120000038; 3120000054; 3120000062
Pipettor tips [2-200 μL, 50-1,000 μL]	Eppendorf	epT.I.P.S. 22491334; 022491351
Scissors
Glass capillaries, 1 x 90 mm GD-1 with filament	Narishige	GD-1
Glass needle puller	Narishige	PN-30
Tubes, Eppendorf	VWR	20170-577
Tube racks (3)	VWR	82024-462
Hot plate	VWR	89090-994
Syringes (10), 30 mL	VWR	CA64000-041
Ultrasound transducer, 20 MHz	VisualSonics Inc.	MS250