Mouse anti repo

Dechorionated embryos were fixed and stained as per Roddie et al.³¹ . Antibodies were diluted in PATx (0.1% Triton-X100 (Sigma-Aldrich), 1% BSA (Sigma-Aldrich) in PBS (Oxoid, Thermo Fisher, MA, USA)). Rabbit anti-GFP (ab290 1:1000; Abcam, Cambridge, UK) or mouse anti-GFP (ab1218 1:200; Abcam) were used to detect GFP-labelled macrophages. Rabbit anti-cDCP-1 (9578S 1:1000; Cell Signaling Technologies), mouse anti-Repo (concentrate of clone 8D12 used at 1:1000; Developmental Studies Hybridoma Bank, University of Iowa, USA) or mouse anti-Futch (supernatant of clone 22C10 used at 1:200; Developmental Studies Hybridoma Bank) were also used as primary antibodies. Goat anti-mouse or goat anti-rabbit secondary antibodies conjugated to AlexaFluor568, AlexaFluor488 (A11036 and A11034; Invitrogen, Thermo Fisher) or FITC (115-095-146; Jackson Immunoresearch, Cambridge, UK) were used to detect primary antibodies; these were diluted from stock solutions made according to the recommendations of the supplier (1:400 in PATx). Stained embryos were stored in DABCO mountant (Sigma-Aldrich) and mounted on slides for imaging.

Larval discs were dissected, fixed and stained followed by protocol as previous described [44 (link)]. Primary antibodies were: mouse-anti Cut (1:200), mouse anti-Repo (1:100), rat-anti-Elav (1:200), rat-anti E-Cadherin (1:25) from Developmental Studies Hybridoma Bank (DSHB, University of Iowa). The anti-cleaved caspase 3 (CC3) antibody [45 (link)] is from Cell Signaling Technology 9661S. Fluorescence conjugated secondary antibodies, including anti-HRP (1:300), were obtained from Jackson ImmunoResearch. Imaging procedures were acquired by LSM 510 Meta or LSM 780 confocal microscope (Zeiss).

The following antibodies were used: Guinea pig anti-β-galactosidase (1:500, a gift from Y. Hiromi), rat anti-β-galactosidase (1:500, a gift from Y. Hiromi), rabbit anti-β-galactosidase (1:500, Cappel), mouse anti-Repo (1:200, Developmental Studies Hybridoma Bank (DSHB) 8D12), mouse anti-Chaoptin (1:100, DSHB, 22B10), rat anti-Miranda (Mira) (1:100), guinea pig anti-Deadpan (Dpn) (1:1000), mouse anti-BrdU (1:250, Becton, Dickinson and Company), mouse anti-Dlp (1:4, DSHB, 13G8), rabbit anti-active caspase 3 (1:500, Abcam), mouse anti-Svp (1:4, a gift from Y. Hiromi), rabbit anti-pH3(1:1000, Upstate Biotechnology), and Alexa Fluor 488, 546, 633 conjugated secondary antibodies (1:500, Invitorgen).
Larval brains were dissected in PBS and fixed with 3.7% paraformaldehyde in PBS for 30 min at room temperature. After washing with PBST (0.1% triton X-100 in PBS), tissues were blocked with 5% normal goat serum in PBST for 30 min. The samples were incubated with primary antibodies at 4 °C overnight. After washing with PBS, the samples were incubated with secondary antibodies for 2 hours at room temperature. The tissues were mounted in Vectashield (VectaLabs) and fluorescent signals were observed by confocal laser scanning microscope (Zeiss LSM710) with a 40X water immersion objective.

Adult brains were dissected in 1× PBS and fixed at room temperature for 25-30 min in 4% formaldehyde in phosphate buffer [4% formaldehyde, 0.1 M phosphate buffer (pH 7.2), 0.2% Triton X-100]. Samples were then washed with 1× PBS and placed in blocking buffer (PBS, 0.2% Triton X-100, 0.1% normal goat serum) for 2 h at room temperature or 4°C overnight. Brains were incubated with primary antibodies diluted with blocking buffer at 4°C overnight, then washed twice with PBST (PBS+0.1% Triton X-100) and incubated in secondary antibodies diluted with blocking buffer for 4 h at room temperature, then washed twice again with PBST. DAPI was added in the final wash for 30 min at room temperature. Brains were mounted on slides in Vectashield (Vector Laboratories, Burlingame, CA). The antibodies used are as follows: rabbit anti-cleaved Dcp-1 (Cell Signaling Technology, Danvers, MA; #9578, 1:100), mouse anti-Repo (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA; 8D12, 1:50), Rat anti-Elav (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA; 7E8A10, 1:250), DAPI (Sigma-Aldrich, St Louis, MO; 1 mg/l), goat anti-mouse Alexa-Fluor-488 (Life Technologies, Carlsbad, CA; 1:200), goat anti-rabbit Alexa-Fluor-568 (Life Technologies, Carlsbad, CA; 1:200), goat anti-rat Alexa-Fluor-633 (Invitrogen, Carlsbad, CA; 1:200).

Brains were dissected, stained, and imaged as described in Sudmeier et al. (2015) (link). The antibodies used are as follows: Chicken anti-Green Fluorescent Protein (Life Technologies, Carlsbad, CA; 1:500), Mouse anti-Repo (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA; 8D12, 1:50), Rat anti-Elav (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA; 7E8A10, 1:250), DAPI (Sigma-Aldrich, St. Louis, MO; 1 mg/L), Goat anti-Chicken Alexa Fluor-488 (Life Technologies, Carlsbad, CA; 1:200), Goat anti-Mouse Alexa Fluor-568 (Life Technologies, Carlsbad, CA; 1:200), and Goat anti-Rat Alexa Fluor-633 (Invitrogen, Carlsbad, CA; 1:200).

Larval discs were dissected, fixed and stained followed by the protocol as previously described^{51 (link)}. Primary antibodies were used: mouse anti-Cut (1:100) and mouse anti-Repo (1:100) from Developmental Studies Hybridoma Bank (DSHB, University of Iowa), Rabbit anti-Phospho H3 (1:500) from Millipore. Goat anti-Htl was a gift from Dr. Christian Klämbt. Fluorescence conjugated secondary antibodies, including anti-HRP, were obtained from Jackson ImmunoResearch. Imaging procedures were acquired by LSM 880 confocal microscope (Zeiss).

GMR-wIR is a White-RNAi driven by a GMR enhancer to reduce the autofluorescence from the retinal pigments. For cryosectioning, adult flies were fixed in 4% paraformaldehyde for 3 h at room temperature. The fly heads with proboscis were removed and incubated in 1x PBS that contained 25% sucrose at 4°C for 24 h and embedded in OCT compound (Tissue-Tek, Sakura). The solidified samples were sliced at a 100-μm thickness using a Leica LX2501 cryostat. The slices were incubated with the following primary antibodies: mouse anti-Repo (1:100), rat anti-Spitz (1:50) (Developmental Studies Hybridoma Bank), rabbit anti-β-Gal (1:500; Cappel), rabbit anti-Cleaved Caspase-3 (Asp175, 1:200, Cell Signaling), rabbit anti-full length Ref(2)P (1:300, a gift from Tor Erik Rusten), and guinea pig anti-Black (1:500, a gift from Bernhard Hovemann) [50 (link)]. The fluorescent secondary antibodies (1:200) were obtained from Jackson ImmunoResearch. DAPI (25 ng/ml, Sigma) was used to stain the DNA and tissue background. Immunolabeled slices were mounted in FocusClear (CelExplorer Labs) and imaged on a Zeiss LSM 510 Meta confocal microscope.