Tetramethylrhodamine conjugated lysine fixable dextran

To evaluate the retinal vascular permeability, 500 μl of PBS containing 100 μg/ml Hoechst stain H33258 (molecular mass, 534 Da; Sigma-Aldrich) and 1 mg/ml tetramethylrhodamine-conjugated lysine-fixable dextran (molecular mass, 10,000 Da; Thermo Fisher Scientific) were injected into the left ventricle according to the procedures described previously7 (link)8 (link)32 (link). After the injection of fluorescent dyes, eyes were enucleated and immediately fixed in 4% paraformaldehyde (PFA) for 15 minutes at room temperature under light protection. Then, retinal flat mounts were prepared as described previously8 (link). They were mounted in fluorescent mounting medium and observed under a Zeiss LSM510 META laser confocal microscope (Carl Zeiss). Experiments were performed independently, at least, 3 times.

To investigate the effect of CypA on blood–brain barrier function in vivo, CypA or its vehicle for negative control was injected intravenously once into mice (200 μg/kg), and the permeability of retinal vasculature was determined 3, 6 and 24 h after the injection of CypA as described previously^{5 (link)–7 (link)}. Briefly, 500 μl of PBS containing 100 μg/ml Hoechst stain H33258 (molecular mass, 534 Da; Sigma-Aldrich) and 1 mg/ml tetramethylrhodamine-conjugated lysine-fixable dextran (molecular mass, 10,000 Da; Thermo Fisher Scientific) were injected into the left ventricle. After the injection of fluorescent dyes, eyes were enucleated and immediately fixed in 4% paraformaldehyde (PFA) for 15 min at room temperature under protection from light, and retinal flat mounts were prepared. They were mounted in fluorescent mounting medium and observed under a Zeiss LSM510 META laser confocal microscope (Carl Zeiss). Experiments were performed independently at least 3 times.