Rhodamine red x donkey anti rabbit

Immunohistochemistry was performed as previously described (Matthews et al., 2007 (link)). Third instar larvae were dissected in 1X PBS, fixed in 4% paraformaldehyde (Electron Microscopy Sciences; CAS #30525-89-4) in PBS for 15 minutes, rinsed three times in PBS-TX (0.3% Triton X-100 in PBS), and blocked for 1 hour at 4°C in normal donkey serum (1:20; Jackson Immunoresearch; RRID: AB_2337258). Primary antibodies used were chicken anti-GFP (1:1000; Abcam; RRID: AB_300798), rabbit anti-DsRed (1:500; Takara Bio, RRID:AB_10013483), goat anti-HRP (1:200; Jackson Immunoresearch; RRID: AB_2338952), mouse anti-Coracle (1:10; Developmental Studies Hybridoma Bank; RRID:AB_1161642, RRID:AB_1161644) and mouse anti-Elav (1:10; Developmental Studies Hybridoma Bank; RRID:AB_528217). Secondary antibodies used were Alexa Fluor 488 donkey anti-chicken (1:200; Jackson Immunoresearch; RRID: AB_2340375), Rhodamine Red-X donkey anti-rabbit (1:200; Jackson Immunoresearch; RRID: AB_2340613), Alexa Fluor 647 donkey anti-goat (1:200; Jackson Immunoresearch; RRID: AB_2340437), Rhodamine Red-X donkey anti-goat (1:200; Jackson Immunoresearch; RRID: AB_2340423), Rhodamine Red-X donkey anti-mouse (1:200; Jackson Immunoresearch; RRID: AB_ AB_2340831), and Alexa Fluor 647 donkey anti-mouse (1:200; Jackson Immunoresearch; RRID: AB_2340862).

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.