Abstract
Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard microscopy techniques, due to the scattering of imaging light by the thick heart tissue, as these vessels are localized deep within the walls of the developing heart. As optical clearing of tissues using organic solvents such as BABB (1 part benzyl alcohol to 2 parts benzyl benzoate) has been shown to greatly improve the optical penetration depth that can be achieved, we combined clearance of whole, PECAM1-immunostained hearts, with laser-scanning confocal microscopy, in order to obtain high-resolution images of vessels throughout the entire heart. BABB clearance of embryonic hearts takes place rapidly and also acts to preserve the fluorescent signal for several weeks; in addition, samples can be imaged multiple times without loss of signal. This straightforward method is also applicable to imaging other types of blood vessels in whole embryos.
Highlights
Establishing a functioning coronary network is crucial for heart function and embryonic development, and analysis of genetic mouse mutants can provide valuable insight into the molecular signals underlying this developmental process
Coronary vessels in whole embryonic hearts were imaged by wholemount immunostaining with anti-PECAM1 antibody followed by optical clearance and confocal microscopy
The straightforward method described here, for the clearance of embryonic mouse hearts with BABB, increases optical penetration and enables the capture of high-resolution images of blood vessels localized in the aorta and ventricular walls
Summary
Establishing a functioning coronary network is crucial for heart function and embryonic development, and analysis of genetic mouse mutants can provide valuable insight into the molecular signals underlying this developmental process. High-resolution imaging of capillaries and arteries throughout the whole depth of the heart is not possible without some form of tissue clearing. Poor optical penetration is caused by the high refractive index of the multiple cellular and extracellular (e.g., collagen and elastic fibers) components of thick tissues. This scatters the imaging light, causing blurring and decreased contrast. We describe a robust and straightforward technique for the BABB clearance of immunostained samples followed by confocal microscopy, with specific reference to the examination of blood vessels in murine hearts from E (embryonic day) 11.5 - 15.5.
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