D1817

10-kDa Tetramethylrhodamine (TMR)-dextran (ThermoFisher # D1817) was dissolved in sterile DPBS at a concentration of 10 mg/ml. TMR dextran was administered as previously described elsewhere [22 (link)]. Following circulation of the dextran tracer brains were harvested in ice-cold DPBS and fixed in 4% PFA overnight. Fixed brains were washed in DPBS the next day, followed by incubation in 30% sucrose for 48 h at 4˚C before embedding in tissue freezing media. 50 μm thick free-floating sections were cut using a Leica cryostat.

Intravital imaging of the mouse ear with intradermal injection has been described previously (Honkura et al., 2018 (link)). Briefly, following systemic administration of 2000 kDa FITC (SigmaAldrich, FD2000S) or TRITC Dextran (ThermoFischer Scientific, D7139) by tail-vein injection, mice were sedated by intraperitoneal injection of Ketamine-Xylazine (120 mg/kg Ketamine, 10 mg/kg Xylazine) and the ear secured to a solid support. Mice were maintained at a body temperature of 37 °C for the entire experiment, maximum 90 min. Time-lapse imaging was performed using single-photon microscopy (Leica SP8). For intradermal EC stimulation, a volume of approximately 0.1 μl histamine (SigmaAldrich, H7125), concentration 10 ng/μl, was injected using a sub-micrometer capillary needle. 10 kDa TRITC Dextran (ThermoFischer Scientific, D1817) was used as a tracer. Leakage sites were identified in time-lapse imaging as defined sites of concentrated dextran in the extravascular space.

Wild-type X. tropicalis embryos were staged to NF stage 3 (4 cell). Using a microinjector, the two dorsally-fated blastomeres were both injected with 2 nL of morpholino mix. Morpholino mix contained a morpholino against meis1 (MO1:8 ng or MO2:16 ng) or against pbx3 (MO1:10 ng or MO2:10 ng) and a labeled dextran tracer (used at 2 mg/mL, ThermoFisher D1817). Embryos were screened at stage 18 for bilateral red fluorescence in the nervous system tissues. Neurulas were raised to stage 41 and used for regeneration assays. Morphant regenerates were collected at 24hpa, 48hpa, and 72hpa for measurement analysis.

To estimate dextran–tetramethylrhodamine leakage, we injected 2 mg/20 g of body weight fluorescence-tagged dextran (D1817; Thermo Fisher Scientific) into the left ventricle and allowed it to cross the intact BBB for 10 min. After perfusion, brains were isolated, fixed, and immersed into a frozen section medium, then treated as described for immunohistochemistry. The presence of fluorescence in the cortical and striatum areas was focused and calculated to quantify BBB leakage.