Rabbit α cc3

Embryo fixation and immunostaining were performed as described (Reuter and Scott, 1990 (link)). The primary antibodies used include rabbit α-SAS (D. Cavener, 1:500), guinea pig α-Sage (1:100), rabbit α-Forkhead (S. Beckendorf,1: 500)1: rat α-CrebA (1:1000), rat α-Pasilla (1:5000), rabbit α-Phospho histone H3 (Abcam, 1:500), rabbit α-CC3 (Cell Signaling, 1:100), α-PKC ζ (C-20, Santa Cruz Biotech,1:500), rat α-DE-Cadherin (DCAD2, DSHB,1:10), mouse α-Coracle (C566.9, DSHB, 1:200), mouse α-alpha-spectrin (3A9, DSHB, 1:1), rabbit α-Laminin (J. Fessler, 1:1000), mouse α-DCSP-2 (6D6, DSHB, 1:200), mouse α-Lamin (ADL84.12, DSHB, 1:200), mouse α-beta-gal (Promega,1:1000) and rabbit α-GFP (Molecular probes, 1:1000). For HRP staining, Biotin-labeled secondary antibodies were used at 1:500 dilution (Molecular Probes). The HRP staining signal was amplified using the Vectastain ABC kit (Vector Labs). For fluorescence staining, Alexa-488- or Alexa-568- or Alexa-647- labeled secondary antibodies were used at a 1:500 dilution (Molecular Probes). Confocal images were obtained using the LSM 700 confocal microscope (Carl Zeiss, Inc.). Whole-mount in situ hybridization was performed as described previously (Lehmann and Tautz, 1994 (link)). Images from in situ hybridized and HRP stained embryos were obtained with an Axiophot microscope (Carl Zeiss, Inc.).

Immunostaining of larval and pupal retinas was performed using standard protocols (Klein, 2008 (link)). Cleaved Caspase 3 (CC3) staining and detection of EdU incorporation were performed as previously described (Meserve and Duronio, 2015 (link)). For EdU pulse labeling of larval discs (such as in Fig. 1C), eye discs were incubated in EdU for 1 hour before fixation. Primary antibodies: 1/1000 rabbit α-PH3 (Millipore 06–570), 1/1000 mouse α-GFP (Abcam ab1218), 1/1000 rabbit α-RFP (Clontech 632496), 1/100 rat α-ELAV (DSHB 9F8A9), 1/100 mouse α-Cut (DSHB 2B10), 1/200 guinea pig α-Sens (H. Bellen), 1/100 rabbit α-CC3 (Cell Signaling 9661). Secondary antibodies (all 1/1000): Oregon Green 488-conjugated goat α-mouse (Invitrogen O6380), Cy3-conjugated goat α-mouse (Jackson ImmunoResearch 115–165-003), Cy5-conjugated goat α-mouse (Jackson ImmunoResearch 115–175-146), Alexa 488-conjugated goat anti-rabbit (Jackson ImmunoResearch 111–545-144), Rhodamine Red-conjugated goat α-rabbit (ThermoFisher Scientific R6394), Cy5-conjugated goat α-rabbit (Abcam ab6564), Cy2-conjugated goat α-rat (Jackson ImmunoResearch 112–225-143), Cy3-conjugated goat α-rat (Jackson ImmunoResearch 112–165-143), Cy5-conjugated goat anti-rat (Jackson ImmunoResearch 712–175-153), Cy5-conjugated donkey α-guinea pig (Jackson ImmunoResearch 706–175-148).