Mw 10000

The permeability assay was as previously described (Yeh et al., 2018 (link)). C135-Gal4; UAS-mCD8GFP females were crossed with UAS-Rho1^dsRNA males at 25°C, and the progeny embryos were raised at 29°C. C135-Gal4; UAS-mCD8GFP flies were used as controls and raised in parallel under the same conditions. Two-day-old adults were used for the permeability assay. FlyNap (triethylamine)-anesthetized adult flies were injected with thin borosilicate needles containing 50 mg/ml tetramethyl-rhodamine dextran (MW 10000, Molecular Probes, #D1816) under a dissecting microscope. Approximately 10 nl of the dye was injected into the soft tissue between the exoskeleton of two abdominal segments of the adult flies. After a 2-h recovery, the eyes of live adult flies were examined and photographed with a Zeiss LSM 880 confocal microscope. Quantification of dye leakage in each eye was measured by the average fluorescence intensity over the whole eye and normalized against the fluorescence intensity of the antenna using Image J.

Two weeks prior to termination of the experiment, descending CST axons were labelled by injecting 1μL of 10% biotinylated dextran amine (BDA) (MW 10 000, Molecular Probes) bilaterally into the sensorimotor cortex at 6 sites per hemisphere. All animals were anaesthetized as for the SCI / cell transplantation procedure and stereotaxic injections were made at a depth of 2 mm dorsoventrally into the sensorimotor cortex region using the following injection coordinates as determined from a microstimulation mapping study [40 ]: in reference to bregma; AP, anterior–posterior; L, lateral) AP −1.5 mm, L 2.5 mm; AP −0.5 mm, L 3.5 mm; AP +0.5 mm, L 3.5 mm; AP +1.0 mm, L 1.5 mm; AP +1.5 mm, L 2.5 mm; AP +2.0 mm, L 3.5 mm.