Norbixin Synthesis and Characterization

All general chemicals were from Sigma (St. Louis, MO, USA). Reagents for cell culture and Alexa Fluor® 488 - conjugated secondary antibodies were from Thermo Fisher Scientific (Waltham, MA, USA). TrueBlack® was from Biotium (Fremont, CA, USA). Goat anti-human Cathepsin D antibody was from Santa Cruz Biotechnology (Dallas, TX, USA), mouse anti-bovine RPE65 antibody and rabbit anti-human GFAP were from Abcam (Cambridge, UK). Ketamine, xylazine, tropicamide and oxybuprocaine chlorhydrate were from Centravet (Maison-Alfort, France). Lubrithal eye gel was from Dechra Pharmaceuticals (Northwich, UK). Optimal cutting temperature compound and other reagents for histology were from Roth Sochiel (Lauterbourg, France). Agar 100 resin kit was from Agar Scientific (Stansted, UK). 9'-cis-Norbixin was prepared from 9'-cis-bixin (AICABIX P, purity 92 %) purchased from Aica-Color (Cusco, Peru) upon alkaline hydrolysis as previously described [22 (link)] and according to Santos et al. [64 (link)]. The obtained product (the 9'-cis isomer) showed an HPLC purity of 97 % as confirmed by ¹H-nuclear magnetic resonance (using malonic acid as internal standard). Fresh solutions of 9'-cis-norbixin, stored as powder at -80°C, were prepared in DMSO.