Immunostaining of whole larval brains was performed as described (47). Briefly, brains were dissected in phosphate-buffered saline (PBS), fixed in 4% formaldehyde, rinsed in PBS–0.3% Triton X-100 (PBST), and blocked for 60 min in PBST with 10% fetal calf serum (PBSTF). Primary and secondary antibodies were incubated in PBSTF overnight at 4°C. Primary antibodies used in this study include rat anti–DE-cadherin [DCAD2, 1:100, Developmental Studies Hybridoma Bank (DSHB)], mouse anti-Dac (mAbdac1-1, 1:100, DSHB), and rabbit anti-H4K16ac (1:1000, Active Motif). We used Alexa Fluor secondary antibodies (1:1000, Life Technologies). DNA was stained with DAPI. Larval brains were mounted in Vectashield (Molecular Probes). Images were acquired with an SP8 Leica confocal image microscope and processed in Adobe Photoshop CS6 and ImageJ.

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