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.

Activation of Notch1 signaling in stromal fibroblasts inhibits melanoma growth by upregulating WISP-1

“…Blood vessel perfusion and laser-scanning confocal microscopy To assess tumor angiogenesis, blood vessels were directly labeled in anesthetized mice by live perfusion using a specially formulated aqueous solution (7 ml per mouse) containing DiI (D-282; Invitrogen), which was administered by direct intracardiac injection prior to killing the animals, as previously reported by Li et al (2008) and Liu et al (2010). A 7-ml volume of fixative (4% paraformaldehyde) was injected following DiI perfusion and the entire tumor tissue was harvested.…”

Characterization of transgene expression and pDNA distribution of the suctioned kidney in mice

“…To visualize blood vessels with transgene expression or pDNA, Clear T2 (Kuwajima et al, 2013) and Scale SQ (Hama et al, 2015) reagents were used. Before immersion in Clear T2 and Scale SQ reagents, mice were perfused with a DiI solution according to the procedure of Li et al (2008). To observe fluorescent-labeled pDNA, mice were perfused after 10 min or 24 h. The cleared kidney was observed by CLSM.…”

“…CUBIC was used to clarify the dispersibility of transgene expression because it allows deep imaging compared with other tissue clearing reagents (Fumoto et al, 2016). Clear T2 and Scale SQ were selected to verify the distribution relationship with blood vessels because they allow staining of blood vessels by carbocyanine dye (Li et al, 2008;Kuwajima et al, 2013;Hama et al, 2015). We have previously demonstrated that pressure as a physical stimulus induces transient cellular membrane permeation and activation of nuclear factor (NF)-jB and activator protein 1 (AP-1) in vivo using the tissue pressure method (Mukai et al, 2010).…”

Immunological Disruption of Antiangiogenic Signals by Recruited Allospecific T Cells Leads to Corneal Allograft Rejection

“…1C). DiI is one of the lipophilic carbocyanine dyes that can incorporate directly into the membrane of endothelial cells, resulting in instantaneous labeling of blood vessels (32). With laser-confocal microscopy, the vessels can be seen clearly in flat-mounted corneas.…”

“…The thoracic cavity was opened, and a blood collection set (23G 3/4) connected to a three-way stopcock luer valve, was inserted through the left ventricle into the ascending aorta, as described (32). Ten milliliters of PBS, followed by 10 ml of 1,19-dioctadecyl-3,3,39,39-tetramethylindocarbocyanine perchlorate (DiI) mixture (Sigma) and then 10 ml 4% paraformaldehyde in PBS were perfused.…”