C1 upright confocal microscope

For each sample, 10–20 larvae were dissected on ice-cold phosphate-buffered saline (PBS) and fixed in 4% formaldehyde in PBS overnight at 4°C. After fixation, the tissue was washed four times (15 min per wash) with 0.3% Triton X-100 in PBS (PBT), then blocked for 30 min at room temperature in 2% heat-inactivated normal donkey serum in PBT. After blocking, the tissue was incubated in a primary antibody solution diluted with 2% heat-inactivated normal donkey serum in PBT overnight at 4°C. We used the guinea pig anti-Senseless (Nolo et al., 2000 (link), 1:1000) and mouse anti-Achaete (Developmental Studies Hybridoma Bank, contributor J. Skeath, supernatant, 1:10) primary antibodies. To compare signal across tissues, we stained for both antigens simultaneously. The washing and blocking procedure was repeated after primary antibody incubation, and then the tissue was incubated in a secondary antibody (1:200 each of anti-guinea pig [Alexa Fluor 546] and anti-mouse [Alexa Fluor 488]) overnight at 4°C. The tissues were washed with PBT and rinsed with PBS, and then the wing imaginal discs were mounted on poly-l-lysine-coated coverslips using Fluoromount-G (SouthernBiotech). Tissues were imaged using either a Leica LSM 510 or a Nikon C1 upright confocal microscope and processed using ImageJ (version 2.0) and Adobe Photoshop CC 2017.