C9100 13 em ccd camera

For bioluminescence recordings, 35 mm FluoroDishes (World Precision Instruments) were prepared with a layer of autoclaved high-vacuum grease (Dow Corning) on the top of the rim and loaded with 1.2 ml recording medium [3.5 g/L DMEM, d-glucose (Sigma-Aldrich), 0.035% sodium bicarbonate, 10 mm HEPES, 1 mg/ml penicillin-streptomycin, 5% B27, and 0.1 mm luciferin in autoclaved Milli-Q water] and a Millicell insert (Millipore) under sterile conditions. Tissue explants were microdissected from slices and placed on the Millicell insert. The dish was sealed by a glass coverslip. Prepared dishes were placed in a 37°C chamber and imaged with a self-contained Olympus LV200 luminescence microscopy system fitted with a cooled Hamamatsu C9100-13 EM-CCD camera using a 20× 0.45 NA LUCPLFLN objective (Olympus). Bright-field (bf) pictures were taken before and after recording of the bioluminescence signal in darkness. This was to reposition the slice following drug treatment and to confirm the regions-of-interest (ROIs). The recording parameters (EM: 4, gain: 1, exposure time: 1800 s) were identical for Fbxl3^+/+ and Fbxl3^Afh/Afh cultures. Treatments were performed by transferring the insert containing the tissue explant to a new culture dish containing fresh media with either vehicle or drug.

Precision-cut ectopic lung slices (275 μm) were prepared as previously described (39 (link)). After washes to remove residual agarose, slices were placed onto cell culture inserts (Millicell) within 35-mm dishes containing 1 ml recording medium and sealed with a coverslip. Dishes were then transferred to a 37°C incubator housing the photomultiplier tubes (PMTs; H6240 MOD1; Hamamatsu Photonics) or placed under a self-contained Olympus LV200 luminescence microscopy system fitted with a cooled Hamamatsu C9100-13 EM-CCD camera (Olympus) as previously described (40 (link)). Individual regions of interest were delineated using ImageJ software (version 1.41o; NIH).