The following primary antibodies were used: rabbit anti-atypical PKC (1:1000) (Santa Cruz Biotechnology), rabbit anti-Asense (Cheng-Yu Lee, Univ. of Michigan, Ann Arbor, MI), rat anti-Dpn (1:100) (Abcam), guinea pig anti-Dpn (1:2000) (Jim Skeath, Washington Univ., St Louis, MO), mouse anti-Elav (1:50) (9F8A9, DSHB), chicken anti-GFP (1:500) (Aves Labs), mouse anti-Lamin (ADL67.10, DSHB), guinea pig anti-Miranda (1:500) (Doe lab), rabbit anti-PH3 (EMD Millipore), mouse anti-Pros purified antibody (1:500) (MR1A, Abcam), and rat anti-Worniu (1:100) (Abcam). Fluorophore-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA). Texas Red™-X Phalloidin (ThermoFisher Scientific) and DAPI (Sigma-Aldrich) were applied after secondary antibody staining by following manufacturer’s protocol. Larvae were raised at 29°C until third instar, and the brains were dissected, fixed and stained by following published procedures (Lai and Doe, 2014 ; Lai et al., 2012 (link)). Images were taken with Zeiss confocal microscope LSM710, processed with open source software Fiji (Schindelin et al., 2012 (link)) and assembled in the software Adobe Photoshop or Illustrator (Adobe Systems Inc., San Jose, CA). All statistical analyses in the figures used one-tail t-test included in Microsoft Excel (Microsoft Corp., Redmond, WA).
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