F view ccd camera

Preparations were observed in a Zeiss Axioplan 2 microscope equipped with appropriate fluorescence filter sets. Black-and-white images were taken either with a cooled F-View CCD camera (DAPI- and CGH-stained preparations) or an Olympus CCD monochrome camera XM10, and captured separately for each fluorescent dye with either AnalySIS software, version 3.2 (Soft Imaging System GmbH) or with cellSens 1.9 digital imaging software (Olympus Europa Holding), respectively. The images were pseudo-coloured and merged using Adobe Photoshop CS4 and CS5 (Adobe Systems Inc.).

Sediment samples and biofilms growing on electrode surface were collected at the end of the treatment “S³” (t = 417 d) for CARD-FISH analysis. 1 g of marine sediment was fixed in formaldehyde and processed as previously reported (Cruz Viggi et al., 2015 (link)). In parallel, microorganisms on the electrode surface were scraped with a sterile spatula, dissolved in PBS buffer with formaldehyde (2% v/v). Microorganisms detached both from the marine sediment particles and from the electrode surface were filtered through 0.2 μm polycarbonate filters (Ø 47 mm, Millipore) by gentle vacuum (<0.2 bar) and stored at −20°C until use. Each sample has been used for Catalyzed Reporter Deposition-Fluorescence In situ Hybridization (CARD-FISH) following the procedure published elsewhere (Matturro et al., 2016a (link)). Oligonucleotide probes targeting Deltaproteobacteria (DELTA495abc; Loy et al., 2002 (link)) and Desulfobulbaceae (DSB706; Schauer et al., 2014 (link)) were employed following the hybridization conditions reported elsewhere (Matturro et al., 2016a (link)). The analysis was performed by epifluorescence microscopy (Olympus, BX51). Images were captured with Olympus F-View CCD camera and handled with Cell^F software (Olympus, Germany).

Two-hundred thousand cells were seeded on cover slips in a 6-well plate (CytoOne, Starlab, UK) to achieve a 40–70 % confluence 24 h later (on the day of fixation). Liquid was aspired and cells covered for 15 min in 2.5 ml 4 % formaldehyde diluted in 37 °C warm PBS. Cells were washed three times with PBS and 60 min blocked in 1 ml blocking buffer (1× PBS, 5 % BSA, 0.3 % Triton-X™ 100). Then 1° antibody anti-Nrf-2 was added 1:100 in antibody dilution buffer (1× PBS, 1 % BSA, 0.3 % Triton-X™ 100) and incubated over night at 4 °C. The cells were then washed three times, each for 5 min with 2 ml PBS, and then incubated with FITC labeled 2° anti rabbit AB for 2 h at room temperature in the dark. The cells were washed three times, each 5 min with 2 ml PBS, again and 300 nM DAPI in PBS added to the cells for 5 min. Then cells were rinsed several times with PBS and finally mounted in PBS, to be viewed under the fluorescence microscope using the appropriate filters. FITC is excited at 485 nm giving an emission of 514 nm. A BX40 fluorescence microscope with an F-View CCD Camera, Cell^F fluorescence imaging software, and oil immersion objective lens (60× magnification) (all from Olympus, Vienna, Austria) were used. DAPI is excited at 358 nm giving an emission of 561 nm. At least three independent biological replicates were performed, each with about 100 cells analyzed.