Immunofluorescence Imaging of Drosophila Tissues

Fly tissues were freshly collected in PBT (PBS with 0.5% Triton X-100) and then fixed in 4% paraformaldehyde (P6148; Sigma-Aldrich) augmented with 0.5% Triton X-100 (X100; Sigma-Aldrich) for 1 h. Then, the fixed samples were incubated with the primary antibody (20 μg/ml for purified mouse monoclonal antibody against DCX-EMAP; Liang et al., 2014 (link)) overnight at 4°C. The next day, the samples were washed six times in PBS (5 min each time) and then incubated in the Alexa-conjugated secondary antibody (A32727; 1:200 dilution; Thermo Fisher Scientific) overnight at 4°C. On the third day, the samples were washed six times in PBS (5 min each time) and imaged using an Andor spinning disk confocal microscope (Andor) equipped with an inverted microscope (IX73; Olympus), an iXon 897 EMCCD, and a 100× UPlanSApo objective (NA 1.40; Olympus) at 25°C. The image acquisition software was Andor IQ 3.0 (Andor).