Rabbit anti apkcζ c20

The following flies were used in this study: brm^T362 is from J Treisman; erm¹, erm², UAS-Erm^CTHA, UAS-Brm (AK Dingwall), 9D11-Gal4 (erm-Gal4; GM Rubin). brm², bap55^LL05905, Erm RNAi (#26778; BDSC) are from Bloomington Drosophila stock center. VDRC RNAi lines used: Brm (GD37720 and 37721GD), Bap60 (KK103634), Snr1 (KK108599, GD12645, and BDRC#32372), Bap55 (GD24704), Moira (GD6969), Bap180 (KK108618), dMi-2 (KK107204), nurf301 (GD46645), Acf1 (GD33446) and ISWI (GD24505). The type II neuroblast driver: w; UAS-Dicer 2, wor-Gal4, ase-Gal80/CyO; UAS-mCD8-GFP/TM3, Ser (Neumuller et al., 2011 (link)).
The primary antibodies used were: guinea-pig anti-Dpn (1:1000, J Skeath), anti-Insc (1:1000); rabbit anti-aPKCζ C20 (1:100; Santa Cruz Biotechnologies, Dallas, TX); guinea-pig anti-Numb (1:1000; J Skeath); mouse anti-Mira (1:50; F Matsuzaki); rat anti-CD8 (1:250; Caltag laboratories, United Kingdom); rabbit anti-GFP (1:500; Molecular Probes, Eugene, OR); rabbit anti-Asense (1:1000; YN Jan); rabbit anti-PntP1 (1:100; J Skeath); rabbit anti-Brm (1:100; L Zhang); rat anti-phospho-Histone H3 (1:1000; Cell Signaling, Danvers, MA); rabbit anti-phospho-Histone H3 (1:200; Sigma, St Louis, MO); mouse anti-dMyc (1:5; B Edgar). Antibodies for western blotting used were: mouse anti-Myc (1:2000; Abcam, United Kingdom) and mouse anti-Flag (1:1000; Sigma).

Third instar larvae were dissected in 1xPBS and fixed in 4% paraformaldehyde (PFA) for 20 min and stained. For dpERK staining, third instar larvae were dissected in cold 1xPBS and immediate fixed in 8% PFA for 20 min and followed with 10 min ice-cold ethanol treatment in −20 °C before further step. Samples were incubated with primary antibodies at the following dilutions: chicken anti-GFP (1:1000, Abcam, ab13970), rabbit anti-phospho-Histone H3 (Ser10) (1:200, Cell signaling, #9701), rabbit anti-Dcp1 (1:100, Cell signaling, #9578), mouse anti-WASH (1:5, Developmental Studies Hybridoma Bank(DSHB), P3H3) mouse anti-MMP1 (1:50, DSHB, cocktail 1:1:1 of 5H7B11, 3B8D12), mouse anti-Ptp10D (1:50, DSHB, cocktail 1:1 of 8B22F5 and 45E10), rabbit anti-aPKCζ (C-20) (1:250, Santa Cruz Biotechnology (SCBT), sc-216,), mouse anti-dEGFR (1:100, Sigma, E2906), guinea pig anti-capicua⁴¹ (1:1000, a kind gift from Edgar’s Lab), Alexa Fluor^TM Phalloidin 647 (1:50, Thermo Fisher, A22287), rabbit anti-ß-galactosidase (1:150, Cappel Laboratories, #0855976), rabbit anti-dpERK (1:200, cell signaling, #4370) guinea pig anti-DIAP1^{59 (link)} (1:200, a kind gift from Meier Pascal’s lab).

Larvae of desired genotype were dissected at 96 h ALH and brains were fixed for 15 min in 3.7% formaldehyde in PBS with 0.1% Triton-X, and later processed for immunochemistry analysis. The following antibodies were used: mouse anti-Mira (Fumio Matsuzaki), 1/30; mouse anti-flag (Sigma), 1/2,000; rabbit anti-Mira49 (link) (generated in our lab), 1/1,000; rabbit anti-Stau (Daniel St Johnston), 1/2,000; rat anti-Brat50 (link) (Yongqing Zhang), 1/500; mouse anti-Pros (DSHB), 1/20; guinea-pig anti-Dpn (generated in our lab), 1/1,000; rabbit anti-Pon (generated in our lab), 1/2,000; rabbit anti-aPKCζ C20 (Santa Cruz Biotechnologies), 1/1,000; chicken anti-GFP (Abcam), 1/5,000. Secondary antibodies were conjugated to Alexa Fluor 488, Alexa Fluor 555, or Alexa Fluor 633 (Molecular Probes), and used at 1/500, 1/1,000, and 1/250, respectively. TO-PRO-3 (Invitrogen) was used at 1/5,000 for DNA staining and samples were mounted in Vectashield (Vector Laboratories). Images were obtained using Zeiss LSM 510 upright or Leica SP II upright microscope and processed in Adobe Photoshop CS6 and Adobe Illustrator CS6.

Ovaries were dissected in PBS, fixed for 20 min in 4% paraformaldehyde (PFA) in PBS, washed for 45 min in PBS/0.1% Triton X‐100 (PBST) and blocked for 30 min in 5% normal goat serum (NGS)/PBST. Primary antibodies were diluted in NGS/PBST, and samples were incubated overnight at 4°C.
Primary antibodies used were FITC‐conjugated anti‐GFP (1:400; Abcam), rat anti‐Crb (1:300, a gift from U.Tepass), anti‐Ex (gift from R. Fehon), rabbit anti‐Kib (1:200, Genevet et al, 2010 (link)), rabbit anti‐aPKCζ (C20) (1:250 Santa Cruz), anti‐pWts (gift from K. Irvine), anti‐Rab5 (DSHB), anti‐Rab7 (DSHB), anti‐Rab11 (DSHB), anti‐alpha‐spec (DSHB). Secondary antibodies (goat Alexa Fluor 488, 546, 647; Invitrogen) were used at 1:500 for 2 h at RT, and samples were mounted in Vectashield (Vector Laboratories).