Gfp booster

U2OS LacO cells (a gift from S Janicki) were grown in DMEM supplemented with 8% FBS (Clontech), hygromycin (200 µg/ml), pen/strep (50 µg/ml), and L-glutamine (2 mM). Cells were transfected with the indicated constructs for 48 hr using Fugene HD according to the manufacturer's protocol. Asynchronously growing cells were arrested in prometaphase by the addition of nocodazole (830 nM) for 2–3 hr. Cells plated on 12-mm coverslips were fixed (with 3.7% paraformaldehyde, 0.1% Triton X-100, 100 mM Pipes, pH 6.8, 1 mM MgCl₂, and 5 mM EGTA) for 5–10 min. Coverslips were washed with PBS and blocked with 3% BSA in PBS for 1 hr, incubated with primary antibodies (GFP-booster [Chromotek], rabbit-anti-BUBR1 [Bethyl] and CREST/anti-centromere antibodies [Cortex Biochem, Inc.]) for 16 hr at 4°C, washed with PBS containing 0.1% Triton X-100, and incubated with secondary antibodies (goat-anti-rabbit Alexa Fluor 568 and goat anti-human Alexa Fluor 647) for an additional hour at room temperature. Coverslips were then washed, incubated with DAPI for 2 min, and mounted using antifade (ProLong; Molecular Probes, Eugene, OR). All images were acquired on a deconvolution system (DeltaVision RT; Applied Precision, part of GE Healthcare) with a 100×/1.40 NA U Plan S Apochromat objective (Olympus, Shinjuku, Tokyo, Japan) using softWoRx software (Applied Precision).

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.

The reporter line for Tim expression was stained with a primary α-GFP antibody from a mouse (DSHB, Iowa City, USA, 8H11) and a goat α-mouse antibody conjugated with Alexa Fluor 488 (Jackson ImmunoResearch, Cambridgeshire, UK, 115-545-062). Sagittal sections of the whole flies were stained with GFP Booster (ChromoTek, Martinsried, Germany, Catalogue number: gb2AF488), because the native GFP fluorescence was impaired during the fixation procedure. Antibody staining of larval and adult tracheae was visualized by an AxioImager Z1 equipped with an Apotome (Carl Zeiss Microscopy, Oberkochen, Germany) and with a Zeiss LSM880 (Carl Zeiss Microscopy, Oberkochen, Germany). To visualize the adult tracheal system, we have utilized the autofluorescence properties of tracheal epithelial cells. Head and thoracic tracheal cells were excited with 405 nm, and detection was conducted at 460 nm. The abdominal tracheal structures were excited with 488 nm and detected with a 516 nm laser. Emitted light was detected using the LSM 880 Airyscan detector with a 458/561 main beam splitter (Carl Zeiss Microscopy, Oberkochen, Germany). Airyscan processed images were selected for maximum intensity projection and digitally stitched together.