Acridine orange staining and immunohistochemistry

Embryos were incubated in 1:5000 acridine orange solution for 1 hr at 28.5°C (stock: 6 mg/ml, Sigma-Aldrich) followed by 2X washes in fish system water. For immunohistochemistry, embryos were processed following standard protocols using 4% PFA fixation, permeabilization using acetone, and incubation in blocking solution for 1 hour. EJC mutant embryos and wild-type siblings at 24 hpf and 26 hpf were incubated in 2% BSA/2% goat serum/1% DMSO/0.1% Tween-20/PBS blocking solution with 1:100 dilution anti-SV2 (DSHB) and 1:1000 anti-A4.1025 (DSHB) primary antibodies and AlexaFlour (Molecular Probes) secondary antibodies. Embryos were stained with Alexa Fluor 488-conjugated α-Bungarotoxin (Thermo Fisher) incubation in a 1:200 blocking solution post primary and secondary antibody staining. All images were centered on the region above the end of the yolk tube which included somites 12–16 at 24 hpf and somites 16–20 at 26 hpf.