Whole-mount Immunofluorescence Assay Protocol

Whole-mount immunofluorescence assay was performed essentially as described previously (Zhang et al., 2012 (link); Xue et al., 2014 (link)). Primary antibodies for immunofluorescence were: mouse anti-GFP (1:200; sc-9996, Santa Cruz), rabbit anti-GFP (1:800; Ab290, Abcam), mouse anti-acetylated tubulin (1:400; T6793, Sigma), rabbit anti-aPKC (1:100; sc-216, Santa Cruz), rabbit anti-pH3 (1:200; #9701, Cell Signaling Technology), rabbit anti-active capase3 (1:200; BD 559565, BD Biosciences), mouse anti-GST (1:400; BE2075, EASYBIO), rabbit anti-p-MLC2 (Thr18/Ser19) (1:200; #3674, Cell Signaling Technology), and rabbit anti-RhoA (1:500; BS1782, Bioworld) antibodies. Secondary antibodies were Alexa Fluor 488- or 649-conjugated anti-mouse IgG (1:100; 115-545-003 and 115-605-003, Invitrogen) and Alexa Fluor 488- or 649-conjugated anti-rabbit IgG (1:100; A11008 and A27040, Invitrogen) antibodies. For imaging, the embryo region containing DFCs or KV was dissected, embedded in the mounting medium and were observed under a Zeiss LSM META confocal microscope. Confocal images of DFCs were acquired as a z-series with a step-size of 2 μm and those of KV as a z-series with a step-size of 1 μm.