Eclipse e800

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.

Samples were imaged using a Nikon Eclipse E800 or Leica DM6000B fluorescence microscope and Zeiss LSM 710 or LSM 510 and Leica SP8 Falcon confocal microscope. Figures were assembled in Adobe Photoshop CS4. Images presented in the figures are raw data images processed for orientation and cropped to size and pixel density with the following exception: In Figure 2 and Supplementary Figures 9, 11, the image brightness was increased to better visualize the data.