Immunolabeling of Drosophila Larval Brains

Third instar larval brains were dissected in PBS and fixed in 100 mM Pipes (pH 6.9), 1 mM EGTA, 0.3% Triton X-100 and 1 mM MgSO₄ containing 4% formaldehyde for 23 min. Fixed brain samples were washed with PBST containing PBS and 0.3% Triton X-100. After removing fix solution, samples were incubated with primary antibodies for 3 h at room temperature. Samples were washed with PBST and incubated with secondary antibodies overnight at 4°C. The next day samples were washed with PBST and then equilibrated in ProLong Gold antifade mountant (ThermoFisher Scientific). The confocal images were acquired on a Leica SP5 scanning confocal microscope (Leica Microsystems Inc, Buffalo Grove, IL). Ten brains per genotype were used to obtain data in each experiment. Antibodies used include rabbit anti-ZLD (1:500) (Harrison et al. 2010 (link)), rat anti-DPN Antibody (1:2) (clone 11D1BC7.14) (Lee et al. 2006 (link)), rabbit anti-ASE (1:400) (Weng et al. 2010 (link)), and chicken anti-GFP Antibody (1:2,000) (Aves Labs, Davis, CA, Cat #GFP-1020).