Puromycin incorporation assay

Puromycin incorporation was assayed using the Click-iT Plus OPP Alexa Fluor Protein Synthesis Assay Kits (ThermoFisher #{"type":"entrez-nucleotide","attrs":{"text":"C10456","term_id":"56146380","term_text":"C10456"}}C10456 and #{"type":"entrez-nucleotide","attrs":{"text":"C10457","term_id":"1535528","term_text":"C10457"}}C10457) according to manufacturer instructions with minor modifications, as outlined below. Larvae were inverted in pre-warmed Schneider’s Drosophila Medium at 25 °C (Gibco, ThermoFisher #21720024) and transferred into a 1.5 ml tube with pre-warmed Schneider’s medium. The medium was then replaced with either 1 μM O-propargyl-puromycin (OPP) in Schneider’s medium for 15 min or 20 μM for 30 min with gentle rotation at room temperature. After OPP incorporation, inverted larvae were rinsed in PBS, fixed in 4 % methanol-free formaldehyde and rinsed again in PBS before a permeabilization and blocking step. The Click-iT reaction mix was prepared as per manufacturer instructions and larvae were incubated for 30 min in the dark at room temperature. Samples were finally rinsed in Reaction Rinse Buffer and stained with DAPI. Discs were dissected out and mounted in mounting medium.