EdU Labeling and Immunostaining in Zebrafish Larvae

For EdU labeling, larvae were incubated in 2.5 mg/ml EdU (VWR) in PBS for 30 min. Larvae were fixed in 4% PFA in PBS overnight at 4°C and rinsed 5× for 20 min in PBTx. Larvae were equilibrated in 100 mM Tris-HCl pH 8 for 2 min and EdU was detected by using a copper-catalyzed azide-alkyne click reaction (25 µM Alexa Fluor 647 Azide (Invitrogen, A10277), 100 mM Tris, 1 mM CuSO4, 100 mM ascorbic acid, pH 8) with 30 min incubation time. Subsequently, larvae were rinsed 5× for 20 min in PBTx. The GFP signal was recovered by using an anti-GFP antibody. Immunostaining was performed as described for anti-pS6, with the following antibodies and dilutions: rabbit anti-GFP antibody (GeneTex, GTX113617; 1:200), goat anti-rabbit antibody conjugated to Alexa Fluor 488 (Invitrogen, A-11070; 1:500). For quantification of EdU⁺ caudal fin mesenchyme, cells within 300 µm anterior of the notochord tip were counted.