Rabbit anti ase

Larva 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 the fix solution, samples were incubated with primary antibodies for 3 h at room temperature. Three hours later, samples were washed with PBST and then incubated with secondary antibodies overnight at 4°C. On the next day, samples were washed with PBST and then equilibrated in ProLong Gold anti-fade mountant (Thermo Fisher Scientific). Antibodies used in this study included chicken anti-GFP (1:2000; Aves Laboratories), rabbit anti-Ase (1:400), rabbit anti-β-gal (1:1000; MP Biomedicals), mouse anti-cMyc (1:200; Sigma), mouse anti-V5 (1:500; Thermo Fisher Scientific), and rat anti-Dpn (1:2). Secondary antibodies were from Jackson ImmunoResearch, Inc.. We used rhodamine phalloidin (Thermo Fisher Scientific) to visualize cortical actin. The confocal images were acquired on a Leica SP5 scanning confocal microscope (Leica Microsystems, Inc). More than 10 brains per genotype were used to obtain data in each experiment.