Whole-mount Immunostaining of Drosophila Larvae

Whole-mount immunostaining was performed as previously described (Choudhury et al., 2016 (link)). In brief, the internal organs of third instar larvae were dissected in 1X PBS (13 mM NaCl, 0.7 mM Na2HPO4, 0.3 mM NaH2PO4, pH 7.4) and fixed in 4% formaldehyde for 20 min at RT. Tissues were washed in 1XPBS followed by 1% Triton X-100 treatment for 20 min. Tissues were washed and incubated in blocking solution (10% Fetal Bovine Serum (FBS), 0.05% Sodium Azide in 1X PBS) for 2 hr at RT and then incubated in primary antibodies at 4°C overnight. Tissues were washed and further incubated with appropriate fluorescent-tagged secondary antibodies for 2 hr, typically. After washing, tissues were incubated in DAPI (4–6-diamidino-2-phenylindole, Sigma-Aldrich, 1 μg/mL) for 10 min and mounted in PromoFluor Antifade Reagent (PK-PF-AFR1, PromoKine) mounting medium and examined using a Leica TCS SP2-AOBS confocal microscope.