Guinea pig anti dpn

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).

Dissected fly tissues were fixed in 4% paraformaldehyde (PFA) in 1× PBS for 10 min at room temperature (RT). The fixative was then removed, and samples were rinsed by 1× PBS and incubated in wash solution (1× PBS + 0.5% horse serum + 0.3% Triton X-100). The samples were incubated in primary antibodies for at least 12 h at RT. They were then briefly rinsed with wash solution and incubated in the DNA dye Hoechst 33342 and secondary antibodies for at least 12 h at RT. Samples were then mounted onto slides for observation under confocal microscopy.
Primary antibodies used in this study included: mouse monoclonal anti-Notch antibody (intracellular domain) C17.9C6 (1:100), mouse monoclonal anti-Repo 8D12 (1:100), mouse monoclonal Abdac1-1 antibody for Dachshund (1:100) from Developmental Studies Hybridoma Bank (DSHB, USA), rabbit anti-CTP synthase y88 (sc-134457, 1:1000 Santa Cruz BioTech, USA); rabbit anti-Prospero (1:1000, a gift from Denan Wang), guinea pig anti-Dpn (1:10000, a gift from James B. Skeath), rabbit anti-DE-cadherin (sc-33743, 1:1000 Santa Cruz BioTech, USA). Secondary antibodies used in this study were anti-mouse, rabbit, guinea pig or goat antibodies labelled with Alexa Fluor^® 488, Cy3 or Cy5 (1:1000, Jackson Immuno Research Laboratories, USA).