O-Propargyl-Puromycin (OPP) Protein Synthesis Assay

Embryos were dissected out of the uterus and placed in a 1:2000 solution of O-propargyl-puromycin (OPP) reagent (Click-iT Plus OPP Alexa Fluor 488, Invitrogen, Carlsbad, CA, United States) in DMEM and left to incubate at 37°C for 30 min. Following this incubation, yolk sacs and amnions were removed to be processed for genotyping while embryos were fixed in 4% PFA for 30 min. After fixation, embryos were stored at 4°C in PBS containing 0.01% sodium azide. Click-it reaction was performed according to the manufacturer’s instructions followed by tissue permeabilization with 1% Triton X-100 in PBS and subsequent 5 min incubation in a 1:50 solution of TrueBlack Lipofuscin Autofluorescence Quencher (Biotium, 23007-F) to diminish autofluorescence. All embryos were imaged at the same magnification (77×), laser power, and exposure time and images were analyzed using ImageJ software to assess fluorescence intensity by measuring the integrated density of the embryo, and subtracting the integrated density of the background. Negative controls to detect and potentially normalize to autofluorescence did either (i) not include OPP reagent during incubation or (ii) omitted click-it reaction. Neither control produced any discernable signal at the illumination and exposure conditions that we used to document outcomes. Thus, OPP outcomes were quantified and directly compared between genotypes. Statistics were performed on GraphPad Prism version 8 using unpaired t-test.