C1 plus confocal microscope

Dissection, staining, and mounting of wandering third-instar larvae were performed as previously described [19 (link)]. For immunohistochemistry (IHC), larvae were dissected in PBS, pinned on Sylgard plates, and fixed in 4% paraformaldehyde for 20 minutes. Next, larvae were washed five times in 1× PBT (PBS + 0.3% Triton X-100). Cuticle filets were blocked for at least 30 minutes at room temperature in 5% normal donkey serum (Jackson Laboratories, West Grove, PA, USA), followed by incubation with respective primary antibodies. Antibody dilutions used were as follows: rabbit anti-Bdwf (1:200), mouse anti-CD8 (1:100) (Invitrogen), rabbit RpS6-(1:100) (Cell Signaling Technology, Danvers, MA, USA), and mouse anti-Cut (2B10; 1:50) (Developmental Studies Hybridoma Bank, Houston, TX, USA). Donkey anti-rabbit and donkey anti-mouse secondary antibodies were used at 1:200 (Jackson Immunoresearch, West Grove, PA, USA). Filets were incubated in glycerol for 5 minutes, followed by being directly mounted onto coverslips with either a drop of glycerol or an aqueous fluoromount. The slides were then imaged on a Nikon C1 Plus confocal microscope or a Zeiss LSM 780.