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Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI

Abstract

We describe a protocol to rapidly and reliably visualize blood vessels in experimental animals. Blood vessels are directly labeled by cardiac perfusion using a specially formulated aqueous solution containing 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), a lipophilic carbocyanine dye, which incorporates into endothelial cell membranes upon contact. By lateral diffusion, DiI also stains membrane structures, including angiogenic sprouts and pseudopodial processes that are not in direct contact. Tissues can be immediately examined by conventional and confocal fluorescence microscopy. High-quality serial optical sections using confocal microscopy are obtainable from thick tissue sections, especially at low magnification, for three-dimensional reconstruction. It takes less than 1 h to stain the vasculature in a whole animal. Compared with alternative techniques to visualize blood vessels, including space-occupying materials such as India ink or fluorescent dye-conjugated dextran, the corrosion casting technique, endothelial cell-specific markers and lectins, the present method simplifies the visualization of blood vessels and data analysis.

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Figure 1: Perfusion device.
Figure 2: Vasculature visualized by DiI labeling.
Figure 3: Three-dimensional reconstructed images of retinal vasculature and angiogenic sprouts and pseudopodial processes.
Figure 4: Penetration of Bruch's membrane by choroidal neovascularization.

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Acknowledgements

We thank Dr George McNamara for critical reading of the manuscript. This work was supported by NIH grant EY12727, EY-015289, P30 EY14801, the Karl Kirchgessner Foundation and the Foundation Fighting Blindness.

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Correspondence to Rong Wen.

Supplementary information

Supplementary Movie 1

Three-D reconstruction of the vasculature of the posterior retina from a mouse eyecup. The clip shows the retinal vasculature rotated around the X-axis. Reconstruction was performed from a stack of confocal images, obtained as described in Fig. 3a, using Leica Application Suite Advanced Fluorescence (LAS AF), 1.7.0, Build 1240 software TCS-SP5, (Leica). (MOV 577 kb)

Supplementary Movie 2

Three-D reconstruction of retinal vasculature from a whole-mount retina. The clip shows an artery and its network rotated around the Y-axis. Reconstruction was performed from a stack of confocal images (obtained with a 10x/0.3 NA objective lens) using software AutoVisualize 3-D (version 5.5, AutoQuant Image, Inc.). See Fig. 3b and text for details. (MOV 1910 kb)

Supplementary Movie 3

Three-D reconstruction of angiogenic sprouts and their pseudopodial processes in a subretinal Matrigel of choroidal neovascularization. The clip shows several sprouts from the choriocapillaris and their pseudopodial processes rotated around the Y-axis. Reconstruction was performed from a stack of confocal images (obtained with a 60x/1.4 NA objective lens) using software AutoVisualize 3-D (version 5.5, AutoQuant Image, Inc.). See Fig. 3c and d for details. (MOV 1019 kb)

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Li, Y., Song, Y., Zhao, L. et al. Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI. Nat Protoc 3, 1703–1708 (2008). https://doi.org/10.1038/nprot.2008.172

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