Application suite las af microscope software

Primary rat cardiac cells were grown on 96-well-plates till 80% confluence, medium was exchanged to non-supplemented DMED/F12 with or without inhibitors. After the treatment, cells were fixed with paraformaldehyde (4%, w/v, in PBS; 15 min), washed (PBS, two times), incubated with CHH (0.2 mmol/L, 2 h, RT), washed (PBS, three times), staining with Nile Red (1:1000, 1 mg/mL in DMSO, 10 min, 37 °C), washed (PBS, three times), and images were acquired using an inverted fluorescence microscope (DMI6000B, Leica Mikrosysteme Vertrieb GmbH, Wetzlar, Germany), equipped with 20 × (NA 0.40) and 40× objectives (NA 0.60), a 12 V/100 W halogen lamp as light source and a Leica DFC360FX camera. Images were acquired with Leica Application Suite (LAS AF) microscope software (version 2.3.0). For Nile Red and CHH images were acquired using λex= 552/λem= 578 and λex= 359/λem= 461, respectively. For confocal fluorescence microscopy experiments cells were cultured on glass-bottom 12-well dishes (Mattek Corporation, Ashland, MA). Images from single confocal sections were acquired every 0.5 µm using Leica SP5 microscope (63× magnification) and the corresponding overlays are shown.