Laminin and poly lysine

For the axonal regrowth analysis after injury, flies were reared at 18°C, in order to minimize overexpression effects during development, and shifted to 25°C the day before injury to allow optimal transgene expression.
Whole-brain explants on culture plate inserts were prepared and injured as described (Ayaz et al., 2008 (link); Koch, 2012 ). In brief, Millicell low height culture plate inserts (Milipore) were coated with laminin and poly-lysine (BD Biosciences). Adult female flies were collected 2–10 days after eclosion and placed on ice. Fly brains were quickly and carefully dissected out in a sterile Petri dish containing ice cold Schneider's Drosophila Medium (GIBCO). Up to seven brains were placed on the membrane of one culture plate insert and culture medium (10,000 U/ml penicillin, 10 mg/ml streptomycin, 10% Foetal Bovine Serum, and 10 μg/ml insulin in Schneider's Drosophila Medium) was added. sLNv axonal injury was performed using an ultrasonic microchisel controlled by a powered device (Eppendorf). Culture dishes were kept in a plastic box in a humidified incubator at 25°C.

To measure axonal outgrowth during development, flies were reared at 25°C and were dissected in fresh PBS. A minimum of 5 fly brains (10 sLNv projections) per genotype were fixed in 4% formaldehyde and stained with an anti-GFP antibody (Molecular Probes; 1:500), according to standard methods (Ayaz et al., 2008 (link)). For the axonal regrowth analysis after injury, flies were reared at 18°C (to minimize developmental effects), and shifted to 25°C 1 day prior to injury. Whole brain explants on culture plate inserts were prepared as described (Ayaz et al., 2008 (link); Koch and Hassan, 2012 (link)). In brief, culture plate inserts (Milipore) were coated with laminin and polylysine (BD Biosciences). Fly brains were carefully dissected out in a sterile Petri dish containing ice cold Schneider's Drosophila Medium (GIBCO), and transferred to one culture plate insert containing culture medium (10 000 U/ml penicillin, 10 mg/ml streptomycin, 10% Fetal Bovine Serum, and 10 μg/ml insulin in Schneider's Drosophila Medium (GIBCO). sLNv axonal injury was performed using an ultrasonic microchisel controlled by a powered device (Eppendorf) as described (Ayaz et al., 2008 (link); Koch and Hassan, 2012 (link)) and dishes were kept in a humidified incubator at 25°C. Four days later, cultured brains were fixed and immunohistochemical staining was performed as for freshly dissected samples.