Dylight 405 donkey anti mouse

Wandering third instar larvae were dissected and pinned open as filets in cold PBS and fixed in 4% paraformaldehyde in PBS for 15 min at room temperature. Larvae were then permeabilized in 0.3% PBST (PBS + Triton X-100) for 15 min at room temperature with agitation, and remaining wash and antibody solutions were made in 0.3% PBST with 1% BSA. Samples were incubated in primary antibody solution overnight at 4°C followed by one quick wash then 5 × 15 min washes using 0.3% PBST before incubating in secondary antibody solution overnight at 4°C. Samples were then washed again as before, then incubated in Vectashield (#H-1000; Vector Labs) for 1 h at room temperature before mounting. Antibodies used were: (1°) anti-Repo (Mouse anti-Repo, #8D12; DSHB), Alexa Fluor 647 anti-HRP (Goat anti-HRP, #123-605-021; Jackson Labs), anti-oaz (Rabbit anti-oaz, (Corty et al., 2022 (link)), anti-GFP (Chicken anti-GFP #ab13970; Abcam); (2°) DyLight 405 Donkey anti-Mouse (#715-475-150; Jackson Labs), Alexa Fluor 488 Donkey anti-Chicken (#703-545-155; Jackson Labs), and Rhodamine Red-X Donkey anti-Rabbit (#711-295-152; Jackson Labs). After staining, larva filets were mounted in Vectashield and covered with #1.5 cover glass (#1404-15; Globe scientific) and stored at 4°C. The same protocol was used for adult wings; however, both primary and secondary incubation steps were done for 5 d at 4°C.