Goat anti mouse igg alexa 568

The primary and conjugated antibodies used in this study are listed in Table S1. Secondary antibodies used for Western blot were goat anti-mouse IgG HRP (BioRad; 1:3000) and polyclonal goat anti-Rabbit IgG HRP (Dako; 1:5000). Secondary antibodies used for immunofluorescence were donkey anti-rabbit IgG Alexa 594 (Invitrogen A21207; 1:400), goat anti-mouse IgG Alexa 568 (Invitrogen A11004; 1:200) and goat anti-guinea pig Alexa 488 (Invitrogen A11073; 1:200). The PP2 and PP3 compounds were purchased from Merck Chemical Ltd. EGF and PDGF-BB were obtained from Sigma-Aldrich. HGF was obtained from R&D Systems and staurosporine was from Tocris Bioscience. The polyclonal rabbit antibodies against the phosphorylated Y1440 and Y1422 sites on β4 are homemade (method described in supplemental materials).

We used immunological stains to identify the positions of proveins in larval wing discs and pupal wings. The primary antibodies used were mouse anti-Delta at 1:50 (Developmental Studies Hybridoma Bank, DSHB), and rat anti-Cubitus interruptus at 1:50 (DSHB). The secondary antibodies were as follows: goat anti-mouse IgG-Alexa 568 and goat anti-rat IgG-Alexa 488 were used at 1:200, were (Invitrogen). Immunostaining was performed as described by Matsuda et al. (2013) (link).

The following primary antibodies were used for immunofluorescence microscopy: human anti-centromere (CREST serum 1:3, Antibodies Inc., 15-234-001; Antibodies Inc., Davis, CA, USA), mouse monoclonal anti-lamin A/C (1:30; Abcam, ab40567; Abcam, Cambridge, UK), monoclonal anti-LAP2α (1:10; clone 15/2, kind gift of Dr. Roland Foisner, Max F. Perutz Laboratories, Vienna, Austria), monoclonal antibody mAb414 (1:2000; Covance, MMS-120R; Covance, Emeryville, CA, USA), monoclonal anti-Hec1 (1:200; clone 3G9, Abcam, ab3613), and rabbit polyclonal anti-LAP2α (1:1000; Abcam, ab5162), polyclonal anti-lamin A (1:500; Sigma-Aldrich, L1293; Sigma-Aldrich, Diegem, Belgium), polyclonal lamin B1 (Abcam, ab16048), polyclonal anti-Sun1 (1:1000, kind gift of Dr. Ulrike Kutay, ETH Zurich, Switzerland), polyclonal anti-Sun2 (1:100; Sigma-Aldrich, HPA001209), polyclonal anti-emerin (1:1000; Bethyl Laboratories, A304-491A; ImTec Diagnostics, Antwerpen, Belgium), as well as polyclonal anti-Nesprin-2 (1:50, kind gift of Dr. Iakowos Karakesisoglou, Durham University, UK). Secondary antibodies were the corresponding goat anti-mouse IgG Alexa 568 (1:1000; Invitrogen), goat anti-mouse IgG Alexa 555 (1:1000; Invitrogen), goat anti-rabbit IgG Alexa 568 (1:1000; Invitrogen), goat anti-mouse IgG Alexa 633 (1:350; Invitrogen), and goat anti-rabbit IgG Alexa 633 (1:350; Invitrogen).

Whole-mount immunostainings of fixed embryos were performed as described previously [67 ]. The following primary antibodies were used: chicken anti-GFP (1:500; Abcam), mouse anti-Spectrin 3A9 (1:10; DSHB), guinea pig anti-Uif (1:500; [68 (link)]), mouse anti-Mega (1:20; [69 (link)]), mouse anti-Crumbs Cq4 (1:50; DSHB), rat anti-DE-cadherin DCAD2 (1:50; DSHB), sheep anti-human-matriptase (1:250; R&D Systems), rabbit anti-human-prostasin (1:250; GeneTex), rabbit anti-human-HAI-2 (1:200; ThermoFisher), and rabbit anti-mCherry (1:500; Rockland). The following secondary antibodies were used in 1:500 dilutions: goat anti-mouse IgG Alexa568, goat anti-mouse IgG Alexa488, goat anti-guinea pig IgG Alexa488, goat anti-rabbit IgG Alexa568, goat anti-rabbit IgG Alexa488 (Invitrogen), goat anti-chicken Alexa488 (Jackson Immuno Research), donkey anti-chicken DyLight549 (Jackson Immuno Research), and donkey anti-sheep Alexa568 (Invitrogen). Fluorescein-conjugated chitin-binding probe (NEB) was used in a 1:500 dilution to stain chitin. Stained embryos were mounted in ProLong Gold antifade reagent (Invitrogen). Image acquisitions were performed with a LSM780 confocal microscope (Zeiss) and a LD LCI Plan-Apochromat 25×/0.8 Imm Corr DIC M27 or a Plan-Apochromat 40×/1.4 Oil DIC M27 oil immersion or a 63×/1.3 Imm Corr DIC M27 LCI Plan-Neofluar (water) objective using standard settings.

Pupal wings were fixed in 3.7% formaldehyde (Sigma-Aldrich) at 4°C overnight. Wing imaginal discs were fixed in 3.7% formaldehyde at room temperature (RT) for 20 minutes. All immunostaining and in situ hybridizations were performed as described previously [7 (link),9 (link)]. The primary antibodies used are as follows: mouse anti-DLG1, rat anti-DE-Cadherin and mouse anti-GFP (for immunohistochemistry; all at 1:50) were obtained from Developmental Studies Hybridoma Bank, rabbit anti-phospho-SMAD1/5 (1: 200 for IF, 1:2000 for Western blotting) from Cell Signaling Technology (CST), rabbit anti-Rab5 (1:600) and rabbit anti-RFP (1:5000 for Western blotting) from Abcam, mouse anti-RFP (1:5000 for Western blotting) from Chromotek, mouse anti-GFP (1: 5000 for Western blotting) from Millipore, mouse anti-β-tubulin (1:5000) from Sigma-Aldrich, rabbit anti-MYC (1:500), goat anti-Scrib (1:100), rabbit anti-aPKC (1:100) and mouse anti-LGL (1:200) from Santa Cruz Biotechnology, and rabbit anti-Scrib (1:2000) from C. Doe. Secondary antibodies were as follows: goat anti-mouse IgG Alexa 488, goat anti-mouse IgG Alexa 568, goat anti-mouse IgG Alexa 647, goat anti-rabbit IgG Alexa 568, goat anti-rabbit IgG Alexa 647, goat anti-rat IgG Alexa 488 and goat anti-mouse IgG Cy5, all from Molecular Probes (1:200). GFP-booster (1:200, ChromoTek) was used to enhance the YFP signal in Fig 3F and S2C Fig.

Fixed embryos were cryoprotected in 20% sucrose overnight at 4°C. Then, the embryos were embedded in 20% sucrose:TissueTek OCT (Sakura, Alphen aan den Rijn, South Holland, Netherlands), sectioned in a cryostat (Leica, CM1850 UV), and collected on gelatin-coated slides. The cryosections were dried, washed with PBS and blocked with 3% NGS (Normal Goat Serum, Jackson Immunoresearch, West Grove, PA, United States) in PBST (PBS with Triton X-100 0.2%, Sigma) for 1h at room temperature in a humid chamber. The primary antibody was diluted in blocking solution and incubated overnight in a humid chamber at room temperature. The primary antibodies used were anti-DsRed2 mouse (1:50; Santa Cruz Biotechnology, cat. Sc-101526), rabbit anti-GFP IgG (1:200; Molecular Probes, cat. A-6455) and mouse anti-HNK-1 IgM (DSHB, cat. 3H5). The secondary antibodies were incubated for 2 h at room temperature and in a humid chamber. The secondary antibodies used were goat anti-mouse IgM coupled to Alexa 488 (1:200, Molecular Probes, cat. A-21042), goat anti-rabbit IgG -Alexa 488 (1:200, Molecular Probes, cat. A-11008), donkey anti-rabbit IgG - Alexa 647 (1:800, Molecular Probes, cat. A31573), goat anti-rabbit IgG–Alexa 568 (1:500, Molecular Probes, cat. A11011) and goat anti-mouse IgG–Alexa 568 (1:500, Molecular Probes, cat. A11004). Finally, the slides were washed and mounted in FluoroShield with DAPI.