Recombinant shh n protein

Intravitreal injections were performed as previously described (Qin et al., 2011 (link); Thomas et al., 2016 (link)). Fish were anesthetized and a small incision was made in the cornea using a Safety Sideport Straight Knife (15°; Beaver-Vistec International). A 33-gauge blunt-end Hamilton Syringe was used to inject 0.5–0.75 microliters of solution. Ouabain injections (10 µM) were performed to damage all retinal neurons as previously described (Fimbel et al., 2007 (link); Sherpa et al., 2014 (link)). Gain- and loss-of-function studies utilized 1X PBS or 1% EtOH for control solutions, and recombinant SHH-N protein (100 µg/mL in 1X PBS; R&D Systems) or cyclopamine (100 µM in 1% EtOH; Toronto Research Chemicals). Light damaged zebrafish were injected beginning at 2 days prior to light onset (− 2dpL) and continued daily through 2 dpL, with amaximum of 5 total injections (Suppl. Fig. 1A). Ouabain damaged retinas were injected beginning at 3 dpi and continued through 10 dpi, with a maximum of 8 total injections (Suppl Fig 1B).

Vismodegib was a gift from Dr. Fred de Sauvage (Genentech). SAG was from EMD Biochemicals. Recombinant ShhN protein was from R&D Systems. Cell Tracker Green CMFDA was from Invitrogen.