Brain and retinal vasculatures exhibit restricted vascular permeability known as blood-brain barrier and blood-retina barrier. Vascular permeability can be evaluated by perfusion of the amine reactive ester derivatives of biotin such as sulfo-NHS-LC-biotin. This protocol describes experimental procedures of sulfo-NHS-LC-biotin perfusion to evaluate retinal vascular permeability. Perfused sulfo-NHS-LC-biotin remained within vessels in wild-type postnatal day 15 (P15) retinas, confirming an intact blood-retina barrier. In contrast, sulfo-NHS-LC-biotin was occasionally detected in extravascular spaces in perfused Eogt^−/− retinas suggesting a partly impaired vascular integrity in the absence of Eogt (Sawaguchi et al., 2017).

Here we provide a detailed protocol for whole mount staining of mouse retina. This protocol was used to analyze retinal angiogenesis in newborn mice (Sawaguchi et al., 2017) by modifying the original protocols (Powner et al., 2012; Tual-Chalot et al., 2013). This protocol can also be used for whole mount staining of adult retina.

This protocol describes how to isolate murine endothelial cells from newborn mice brain and 3-month-old mice lung by modifying the original protocols (Sobczak et al., 2010; Ruck et al., 2014). We have used the protocol to analyze mRNA expression level in brain endothelial cells (Sawaguchi et al., 2017). Isolated lung endothelial cells were expanded in vitro for various downstream experiments such as gene expression analysis and cell-based signaling assay.

This protocol describes how to measure interaction between Notch receptors and their ligands by cell-based assay using Dynabeads. We have used the protocol to determine binding capacity between Notch1-transfected HEK293T cells and ligand-coated Dynabeads. Expression of Eogt in Notch1-expressing cells promoted binding toward DLL4-coated beads, but not JAG1-coated beads. The Notch-ligand assay using Dynabeads suggested that Eogt facilitates DLL4-Notch1 interaction (Sawaguchi et al., 2017).