Lsm 510 meta confocal microscope

Ovaries and discs were dissected in PBS, fixed in 4% PFA for 20 minutes at room temperature and then rinsed three times in phosphate buffered saline with 0,1% Triton X-100. To increase permeabilization of the antibody in the tissue, ovaries have been treated for 10 min with 1% triton X-100. Before incubation with primary antibody ovaries and discs have been incubated with a blocking solution composed of 5% BSA in PBS-Triton 0,1%. Primary antibodies were used for immunostaining against the following antigens: Hnt, Cut, Notch ECD, Notch ICD, (all from Developmental Studies Hybridoma Bank- DSHB); Dome (A gift from Stephane Noselli). Avl (Lu and Bilder, 2005); Ubiquitin FK2 (Biomol); activated Caspase-3 (Signal Transduction Technologies). Secondary antibodies conjugated to Alexa-488, Alex-568 were used (Molecular Probes). Phallodin-TRITC from sigma was used to mark F-actin while DAPI (4′6-diamidino-2-phenylindole) to stain the nuclei. The images were obtained using a Zeiss LSM 510-Meta confocal microscope or aa TCS microscope (Leica). Images were edited with Adobe Photoshop CS and were assembled with Adobe Illustrator.

Imaginal discs to be imaged by confocal microscopy were mounted in Vectashield mounting media with DAPI (Vector Labs). Confocal images were taken with a Zeiss LSM510 Meta confocal microscope or a Leica TCS SP8 STEAD 3X confocal microscope with 405nm, 488nm, 561nm, and 633nm lasers. Both microscopes gave similar results. Measurements of disc size were performed from images of at least fifteen discs using NIH Image-J software.

Cells were imaged 48–72 h posttransfection in phosphate-buffered saline at 37°C, 95% air-5% CO₂, supplemented with glucose and/or inhibitors using a Zeiss LSM 510 Meta confocal microscope with a ×20 Plan-Neofluar lens or a Leica SP8 with a ×20 PL APO CS2 lens. FLII12Pglu-700µΔ6 contains the FRET paired fluorophores enhanced CFP (eCFP; donor) and Citrine (acceptor), which report a reduced eCFP-to-Citrine FRET ratio with a binding of glucose. This was measured on the Zeiss LSM 510 by collecting emission data from eCFP (459–505 nm) and Citrine (525–600 nm) every 4 s over an 8-min time period while exciting eCFP at 458 nm. Settings were optimized for the growth conditions of each cell type, which took into account opacity of the substrate (i.e., glass coverslips and Transwells), cell height, and cell density. Thus, the output measurement was different for the three conditions studied.