Mouse anti β galactosidase

The following antibodies were obtained from commercial sources: rabbit anti-green fluorescent protein (GFP; catalog no. 598, Medical and Biological Laboratories, Woburn, MA, USA), rabbit anti-S6 ribosomal protein (catalog no. 2217, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-S6 (Ser-240/244, catalog no. 2215, Cell Signaling Technology), rabbit anti-phospho-Akt (Ser-473, catalog no. 4060, Cell Signaling Technology), mouse anti-Akt (catalog no. 2920, Cell Signaling Technology), mouse anti-β-galactosidase (catalog no. Z3781, Promega, Madison, WI, USA), rabbit anti-β-galactosidase (catalog no. 559762, MP Biomedicals, Santa Ana, CA, USA), mouse anti-monoubiquitinylated and polyubiquitinylated conjugates (clone FK; catalog no. BML-PW8810, Enzo Life Sciences, Farmingdale, NY, USA), and mouse anti-tubulin (catalog no. T5168, Sigma-Aldrich, St. Louis, MO, USA). Anti-mouse and anti-rabbit secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 647 were obtained from Invitrogen (Carlsbad, CA, USA). IRDye-conjugated secondary donkey anti-mouse IRDye 680RD antibody (catalog no. 926-68072) and donkey anti-rabbit IRDye 800CW antibody (catalog no. 926-32213) were obtained from LI-COR Biosciences (Lincoln, NE, USA). Insulin was obtained from Sigma-Aldrich (St. Louis, MO, USA).

Third-instar larvae were dissected in phosphate-buffered saline (PBS). Wing imaginal discs were fixed in 4% paraformaldehyde at room temperature for 20 min and then washed three times for 5 min in PBS. Discs were blocked [2% BSA diluted in PBS 0.1% Triton X-100 (PBST)] for 45 min at room temperature, followed by incubation with primary antibodies (diluted in block) overnight at 4°C. Tissue was then washed three times in PBST and incubated with secondary antibodies (diluted in block) at room temperature for 1.5 h. Tissue was then washed a final three times in PBST and mounted in a 70% glycerol solution. The following primary antibodies and dilutions were used: mouse anti-β-galactosidase (1:2000, Promega, Madison, USA), mouse anti-Wg [1:100, Developmental Studies Hybridoma Bank (DSHB), Iowa City, USA], mouse anti-Cut (1:50 DSHB), mouse anti-N^ICD (1:50 DSHB), rat anti-Ci (1:50 DSHB), mouse anti-Delta (1:50 DSHB), mouse anti-Arm (1:50 DSHB), rabbit anti-cleaved Caspase 3 (1:100 Cell Signaling), rabbit anti-PH3 (1:200 Cell Signaling), guinea pig anti-Sens (1:500, a gift from Hugo Bellen, Dept. of Molecular and Human Genetics, Baylor College of Medicine, Houston, USA), mouse anti-Dll (1:300, a gift from Ian Duncan, Dept. of Biology, Washington University in St. Louis, St. Louis, USA). Secondary antibodies (Jackson ImmunoResearch, West Grove, USA) were used at a 1:200 dilution.

The genotype of the animal shown in Fig. 8 was y w hsFlp/Y; ex⁶⁹⁷FRT42D ubi-GFP/FRT42D, hpo42 (link)43 (link)44 (link)45 (link)46 (link)47 (link); hh-Gal4/msn-RNAi. The msn-RNAi line was the verified TRiP line BSC2879146 (link). Wandering third instar larvae of this genotype were dissected in cold 1 × phosphate-buffered saline (PBS; pH 7.2) and fixed in 4% formaldehyde in 1 × PBS for 50 min. Samples were permeabilized in 1 × PBT (1 × PBS+0.3% Triton X-100) and blocked in 5% normal donkey serum in PBT. Primary antibodies were incubated overnight at 4 °C: mouse anti-β-galactosidase (Promega, 1:2,000) and rat anti-Cubitus interruptus (DSHB, 1:150). Secondary antibodies were incubated for 3 h at room temperature: donkey anti-mouse Cy3 (Jackson, 1:600) and donkey anti-rat Cy5 (Jackson, 1:600). Samples were mounted in Vectashield (Vector Labs) and imaged using an Olympus FV1000. Images were processed using ImageJ, and figures prepared using Adobe software.

Antibodies and dilutions used were mouse anti-Ptc antibody 1:50 (DSHB, Apa1; 1/50); rat anti-Ci antibody 1:50 (DSHB, 2A1; 1/50); rabbit anti-Dpp prodomain (Akiyama and Gibson, 2015 (link); 1/100); rabbit anti-GFP (Invitrogen, A-11122; 1/1000); chicken anti-GFP (Abcam Cat# ab13970; 1/2000); rabbit anti-phospho-Smad1/5 (Ser463/465) (Cell Signaling Technology, 9516; 1/100); mouse anti-HA.11 (Covance, Cat#101P; 1/1000); rabbit anti-HA (Thermo Fisher Scientific, Cat# 71-5500; 1/1000); mouse anti-β-tubulin (DSHB, E7; 1/5000); mouse anti-β-galactosidase (Promega, Z378A; 1/100); mouse anti-Myc (Santa Cruz Biotechnology, 9E10; 1/200); mouse anti-FLAG (Sigma-Aldrich, M2; 1/200); rabbit anti-FLAG (Sigma-Aldrich, Cat# F7425; 1/200); HRP-conjugated and fluorophore-conjugated secondary antibodies were from Jackson ImmunoResearch Lab and Thermo Fisher. The antibody information is also listed in the Key resources table.

Primary antibodies used for immunohistochemistry were: Mouse anti-β-galactosidase (1:5000; ref 18637314, Promega), rabbit anti-TRPV1 (1:1000, ref sc-28759, Santa Cruz), rabbit anti-CGRP (1:1000, ref AB15360, Abcam), rabbit anti-substance P (1:500, ref 84500505, Biogenesis Ltd), rabbit anti-TrkA (1:500, ref SAB1305371, Sigma), goat anti-c-ret (1:500; ref AF482, R&D System), rabbit anti-phospho GluN1 (Ser896, 1:1000; Upstate), rabbit anti-phospho-Erk (1:1000, ref 9101, Cell Signalling), rabbit anti-HA (1:500, ref 715500, Life Technologies), mouse anti-Iba1 (1:2000, ref MNK4428, Wako), Griffonia simplicifolia isolectin (IB4) FITC conjugated (1:1000, Sigma). Secondary antibodies were Alexa Fluor 488 anti-rabbit or anti-goat antibody (1:2000) from Life Technologies, and Cy3-conjugated anti-mouse antibody (1:2000) was from Jackson ImmunoResearch Laboratories.