Dc350 fx

At the end of each experiment, mice were sacrificed with a lethal dose of sodium pentobarbital and perfused with a solution containing 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). Brains were extracted and stored in PBS solution until the cutting. Before cutting, brains were transferred into PBS containing 30% sucrose for at least 48 hr. Coronal slices (25 μm thick) containing the entire striatum from the recorded side (from AP 1.4 mm to AP −1.3 mm, following Paxinos and Franklin, 2001 ), were obtained using a digital automatic cryotome and collected on gelatine coated slides. Sections were incubated over night with Cy3-conjugated streptavidin (Jackson Immuno Research Laboratories) diluted (1:1000) in 1% BSA, 0.3% Triton-X 100 in 0.1 M PBS. Finally, the glass slides were covered with mowiol (Calbiochem) and imaged. Neurons were then reconstructed using a fluorescence microscope (DM 6000B, Leica) and a camera (DC350 FX, Leica) and then processed by ImageJ.

Stock solutions of Hoechst 33342 (10 mg/mL in H₂O) and PI (1 mg/mL in H₂O) were prepared with final concentrations of PI (0.65 mg/mL) and of Hoechst 33342 (7.5 mg/mL). Cultures were incubated with each substance for 5 min, washed and observed in serum-free supplemented DMEM medium. Apoptotic/necrotic cells were detected by the uptake of PI into cell nuclei, since vital cells did not incorporate PI. In contrast, Hoechst 33342 was incorporated into nuclei of all cells. Differentiation of necrosis or apoptosis was not possible with this method. This ratio demonstrates the ratio of dying cells to all cells and was declared in percent. Images were taken at 100 × magnification with a computer-driven digital camera (Leica DC350 FX,Wetzlar, Germany) on an inverted microscope (Leica DM IRE2 HC FLUO, Wentzler, Germany) equipped with an incubator and temperature control for live cell experiments. Red fluorescence (PI) was visualized with the TRITC filter [excitation 515–560 Band Pass (BP)/emission 590 Long Pass] and blue fluorescence (Hoechst 33342) with the DAPI filter [excitation 340–380 Band Pass (BP)/emission 425 Long Pass]. LEICA FW4000-I was used as software. Image analysis was performed densitometrically with ADOBE PHOTOSHOP (Munich, Germany).

In order to establish the potential oxidative damage to cells and tissues, histochemical analyses with diaminobenzidine (DAB) and dichlorodihydrofluoresce in diacetate (DCFDA-SIGMA D6883) were performed. For DAB staining assays, small thallus sections of 1 mm to 5 mm were made and the samples were placed in DAB (pH 7.0, 0.5 g L⁻¹) overnight, then dehydrated in 100% ethanol and observed under a light optical microscope. The qualitative damage was observed by the presence of DCF oxidised under a Nikon Eclipse E800 fluorescence microscope with a Leica DC 350FX camera. The emission of DCF oxidised registered in the range of 530 nm using a Synergy™ HTX Multi-Mode Microplate Reader; fluorescence was measured in terms of the intensity of the fluorescence emitted [62 (link)]. DCF fluorescence intensity was recorded using plants discs of 5 mm in diameter. Plants grown without the contaminant, but with and without DCFDA, were used as control treatments. The plant discs of each treatment were placed in 96-well microplates and excited at 485 nm. An overlap showing the fluorescence of both chlorophyll and DCF was made without the red light filter to observe the specific location of the ROS.