Tbuoh dmso

NHS-Azide-injected samples were processed for click chemistry according to the following protocol, modified from previous studies (Dieterich et al., 2010 (link); Hinz et al., 2012 (link)). Sections or wholemounts were transferred to Eppendorf tubes or six-well plates with reaction mixture composed of 100 μm tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA, Sigma) dissolved in 4:1 tBuOH/DMSO (Sigma), 100 μm CuSO₄ (Sigma), 1.25 μm Alexa Fluor 488 alkyne (Invitrogen), and 250 μm tris(2-carboxyethyl)phosphine (TCEP, Sigma). The reaction proceeded overnight at room temperature.

500 mM AHA (L-azidohomoalanine, 500 mM, pH7.4, Clickchemistry tools) colored with ~0.01% fast green was injected into the tectal ventricle of anesthetized stage 47/48 tadpoles. Animals recovered from anesthesia for 0.5 hr and then were exposed to VE for 0.5 hr followed by 4 hr ambient light before dissecting out their midbrains. We dissected midbrains from 1200 to 1500 stage 47/48 tadpoles for each experimental group in order to yield about 15 mg protein in two separate experiments. Brains were homogenized in phosphate-buffered saline (PBS) containing 0.5% SDS and protease inhibitors (PI; Roche, complete ULTRA Tablets, Mini, EDTA-free Protease Inhibitor cocktail tablets) followed by sonication with a probe sonicator. Samples were boiled for 5 min and small aliquots were taken to measure protein concentration using the BCA Protein Assay Kit (Thermo Fisher Scientific, 23227). 5–10 µg of the sample was saved as total protein sample, while the rest was used for the following click reaction. For each 400 µL reaction, 1.5 mg of total protein was used with 1.7 mM Triazole ligand (Invitrogen) in 4:1 tBuOH/DMSO (Sigma), 50 mM CuSO4 (Sigma), 5 mM Biotin Alkyne (Invitrogen) and 50 mM TCEP (Sigma) added in sequence. The reaction proceeded for 1–2 hr at room temperature. Excess reagents were removed with methanol/chloroform/water precipitation.