Improved vessel painting with carbocyanine dye-liposome solution for visualisation of vasculature

Alu Konno,Shigetoshi Okazaki,Naoya Matsumoto

doi:10.1038/s41598-017-09496-4

Alu Konno, Shigetoshi Okazaki + Show 1 more

Open Access

https://doi.org/10.1038/s41598-017-09496-4

Copy DOI

Abstract

Vessel painting is one of the most accessible and cost-effective techniques for visualising vasculature by fluorescence microscopy. In this method, the hydrophobic carbocyanine dye DiIC18 labels the plasma membrane via insertion of its alkyl chains into the lipid bilayer. A major disadvantage of this procedure is that it does not stain veins and some microvessels in mouse brain. Furthermore, DiIC18 molecules can aggregate during perfusion, thereby occluding arteries and reducing the success rate and reproducibility of the experiment. To overcome these problems, we developed an improved vessel painting procedure that employs neutral liposomes (NLs) and DiIC12. NLs prevented DiI aggregation under physiological conditions whereas DiIC12 showed enhanced dye incorporation into liposomes and consequently increased staining intensity. Using this method, we successfully labelled all major blood vessel types in the mouse brain, including both veins and microvessels. Thus, liposome-mediated vessel painting is a simple and efficient method for visualising vasculature.

Highlights

The cardiovascular system is a continuous network that circulates oxygen, nutrients, signalling molecules, and cells and removes metabolic waste
We developed an improved vessel painting method using neutral liposomes (NLs) and 1,1′-didodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiIC12) to achieve brighter labelling of all major blood vessel types in mouse brain
We speculated that incorporation of DiIC18 molecules into liposomal membranes would lead to their dispersal during vessel painting

Summary

Introduction

The cardiovascular system is a continuous network that circulates oxygen, nutrients, signalling molecules, and cells and removes metabolic waste. Several methods have been developed to visualise vasculature by fluorescence microscopy[2], including transgene expression or immunodetection of vascular endothelial markers[3] and infusion of fluorescent dextrans[4, 5], labelled lectins[6], Alexa Fluor 633 hydrazide[7], or fluorescent resin[8]. Highly hydrophobic DiIC18 molecules form aggregates during perfusion and occlude microvessels, leading to heterogeneous labelling. These challenges must be overcome in order to clearly detect and identify each element of vasculature. To this end, we developed an improved vessel painting method using neutral liposomes (NLs) and 1,1′-didodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiIC12) to achieve brighter labelling of all major blood vessel types in mouse brain. Our results demonstrate that this procedure is useful for visualising even the smallest vascular elements in the brain

Methods

Results

Conclusion