Anti dlg

Wandering third-instar larvae of both sexes from various genotypes were dissected and fixed in Bouin’s fixative for 15 min and processed for immunohistochemistry as previously described (Chen et al., 2012 (link)). Confocal images of all genotypes of larvae belonging to the same experimental group were acquired using the same settings with a Zeiss LSM710 confocal microscope and image editing was done using Adobe Photoshop. Primary antibodies used were FITC-conjugated anti-HRP (1:250, Jackson ImmunoResearch Laboratories), guinea pig anti-Ringer (1:250, Mino et al., 2016 (link)), anti-Ac-Tub (1:1000, T7451, Sigma), anti-Tubulin (1:1000, 2144S, Cell Signaling), and mouse monoclonal anti-Dlg (1:1000, 4F3), anti-Brp (1:250; NC82), anti-Futsch (1:1000; 22C10), and anti-GluR IIA (1:250, 8B4D2) were obtained from Developmental Studies Hybridoma Bank (DSHB), University of Iowa. Secondary antibodies conjugated to Alexa 488 and 568 (Invitrogen-Molecular Probes) were used at 1:400 dilution.

Larval, pupal, and adult brains were rapidly dissected in PBS and fixed in 4% formaldehyde (Polysciences, Inc), larval for 15 min and pupal-adult for 30 min. Staining was performed as described (Kucherenko et al., 2012 (link)). Samples were mounted in 70% glycerol. The following antibodies were used: polyclonal rabbit anti-Dg 1:1000 (Deng et al., 2003 (link)), polyclonal chicken anti-GFP 1:2000 (Invitrogen), monoclonal mouse anti-Sec5 1:50 (gift from Thomas Schwarz [Langevin et al., 2005 (link)]), and anti-FasII 1:20, anti-Dlg 1:20, anti-Elav 1:20, anti-Arm 1:50, anti-FasII 1:50, anti-αPS2 1:50, and rat anti-DE-cadherin 1:50 from Developmental Studies Hybridoma Bank. Alexa 488, 568, goat, anti-rabbit, and anti-chicken 1:500 (Molecular Probes). To visualize nuclei, a 10-min-long incubation with 1× DAPI (Sigma Aldrich) in PBS was performed.

Nota of staged pupae fixed as previously described (Zitserman and Roegiers, 2011 (link)) (see supplementary Materials and Methods for further details). Primary antibodies were anti-GFP (1:1000; Abcam) and anti-Dlg (1:500; Developmental Studies Hybridoma Bank). Secondary antibodies were Alexa Fluor 488-conjugated anti-chicken and Alexa Fluor 568-conjugated anti-mouse (both 1:1000; Thermo Fisher Scientific). EdU staining was performed using the Click-iT EdU Imaging Kit (Thermo Fisher Scientific).

Standard fixation and staining protocols were used on dissected third instar larva eye imaginal discs. Briefly, after dissection on ice-cold PBS, fixation using 4% paraformaldehyde was performed. Washes and permeabilization were carried out using 0.1% Triton X-100. Blocking was then performed for 30 min using bovine serum albumin (0.1%). Primary antibodies used for incubation overnight were anti-Sens (guinea pig 1:2,000, obtained from H. Bellen), anti-FasIII (mouse monoclonal 1:20, Developmental Studies Hybridoma Bank), anti-Dlg (mouse monoclonal 1:100, Developmental Studies Hybridoma Bank), anti-GFP (chick 1:2,000, Aves Labs) and anti-Dcp-1 (rabbit 1:100, Cell Signaling Technology). Secondary antibodies used for 2-h incubation were anti-guinea pig Alexa 647 (1:800), anti-mouse Alexa 488 (1:800), anti-rabbit Alexa 488 (1:800), anti-mouse Alexa 555 (1:800) and anti-chick Dy-Light (1:800), all obtained from Molecular Probes.

Unless otherwise stated, accessory glands from 5 to 6‐day‐old males were dissected in ice‐cold Grace's Insect Medium (BioConcept), fixed for 20 minutes with 4% Formaldehyde (Sigma) at room temperature and stained with one or more of the following antibodies over‐night at 4°C: anti‐Dlg (Developmental Studies Hybridoma Bank [DHSB]), anti‐DE‐cadherin (DHSB), anti‐Rab7 (DHSB) or with Phalloidin‐546 (Life Technologies). Rabbit polyclonal antibodies against CG1656 (1:500) and CG17575 (1:250) were kindly provided by Mariana Wolfner (Cornell University).86 All samples were mounted in Vectashield mounting medium with or without DAPI (Vector Labs). The pictures were taken with a Zeiss LSM700 confocal microscope and evaluated using the FIJI97 (Laboratory of Optical and Computational Instrumentation [LOCI], University of Wisconsin‐Madison) and IMARIS softwares (Bitplane AG).

Embryos were fixed 20 minutes in a mix formaldehyde 4%/heptane, after removing the heptane, MetOH was added and the vitelline membrane removed by vigorous shaking.
The following primary antibodies were used: guinea pig anti-Scrib (a gift from D. Bilder), mouse anti-Myc (Cell Signalling 1:500), rabbit anti-GFP (Molecular Probes, 1:300), mouse anti-β-gal (Cappel, 1:1.000), mouse anti-aPKC (Santa Cruz Biotechnology 1:100), anti-Dlg (1:100) from Developmental Studies HybridomaBank, Phalloidin-Rhodamine (Molecular Probes). Secondary antibodies were coupled to Alexa488, Alexa555 or Alexa647 (Molecular Probes).
Images were taken on an SP2-AOBS or an SPE Leica confocal microscopes and processed using FIJI and Adobe Photoshop programs. For the higher resolution images in Suppl. Figure 2, a Zeiss LSM 880 Laser Scanning Microscope with Airyscan was used.

Flies were dissected in Grace's media and ovaries were fixed and stained as described previously (Tanner et al., 2011 (link)). Samples were mounted in VectaShield with DAPI (Vector Labs). Primary antibodies used were: anti-cleaved-Dcp-1 (1:100, Cell Signaling Technology), anti-Dlg [1:100, Developmental Studies Hybridoma Bank (DSHB)], anti-αPS3 (1:300, Shigeo Hayashi, or 1:1000), anti-βPS (1:10, DSHB), anti-Drpr (1:50, DSHB 5D14), anti-β-Gal (1:400, Promega), anti-aPKC (1:1000, Santa Cruz Biotechnology, Inc.), anti-Dynein (1:3, DSHB 2C11), anti-Kinesin (1:100, Cytoskeleton, Inc.), anti-Crumbs (1:25, DSHB Cq4, protocol from Tanentzapf et al., 2000 (link)) and anti-Talin (mixture of A22A and E16B, 1:50 each, DSHB). The anti-αPS3 antibody was made using the peptide sequence utilized for the original antibody (Wada et al., 2007 (link)) and generated by YenZym (San Francisco, CA). The serum was affinity-purified twice before use. It shows the same expression pattern as the original antibody from the Hayashi lab. Secondary antibodies used were goat-anti-rabbit Cy3, goat-anti-mouse Cy3, goat-anti-mouse Alexa Fluor 647 (Jackson ImmunoResearch), each at 1:100, and goat-anti-rabbit Alexa Fluor 488 (Invitrogen) at 1:200. Egg chambers were imaged on an Olympus FV10i confocal microscope, images were processed using ImageJ and Adobe Photoshop, and figures were made using Adobe Illustrator.