Zen black 2012 black edition software

Trophozoites were stained with primary polyclonal antibody against β-actin (1:1000; ab8227, Abcam, Cambridge, UK) incubated for 1 h, then washed with PBS and further incubated with secondary antibody Alexa Fluor 488 conjugated goat anti rabbit-IgG (H+L) (1:1000; A-11008, Invitrogen, Eugene, Oregon, USA). Then 6 μM (1 UI/ml) rhodamine-phalloidin (Molecular probes, Eugene, Oregon, USA) were incubated for 30 min and Hoechst 33342 for 10 min for nuclear staining. Before this step, trophozoites were treated with ACh 1, 0.01, 0.0001, 0.000001, or 1 µM cytochalasin D (CD, Thermo Fisher Scientific, Waltham, Massachusetts, USA), then fixed with 2% PFA and permeabilized with 0.2% Triton X-100 in PBS. Finally, slides were mounted in Mowiol (Sigma-Aldrich, St Louis, Missouri, USA) and examined by confocal microscopy using Carl Zeiss LSM 700 Laser Scanning Microscope (Carl Zeiss AG) with a 63× oil immersion objective. Images were acquired using the Zen Black 2012 (black edition) software (ZEISS). Quantification of fluorescence intensity of fibrillar actin (F-actin) and globular actin (G-actin) structures was performed with the ImageJ software (Wayne Rasband, Nat. Inst. of Health, USA). Images were converted to 8-bit greyscale and mean fluorescence intensity was measured and expressed as the mean grey value, that is defined as the average grey value for all pixels within the indicated area.

In order to study the morphology of mono and two-species biofilms, E. coli biofilms with or without A. pleuropneumoniae 719 were prepared as described above and stained with FilmTracer FM 1-43 (Invitrogen, Eugene, OR), Wheat Germ Agglutinin (WGA-Oregon Green 488, Molecular Probes), Film Tracer TM SYPRO® Ruby biofilm matrix stain (Molecular Probes), or BOBOTM-3 iodide (Molecular Probes) according to manufacturer's instructions (fluorescent markers stain bacterial membranes, N-acetyl-Dglucosamine [PGA] and N-acetylneuraminic acid residues, proteins and extracellular DNA or eDNA, respectively). After 30 min of incubation at room temperature, the fluorescent marker solution was removed, and the biofilms were washed with water. After that, the biofilms were observed by confocal laser scanning microscopy (CLSM; LMS 700 ZEISS; Carl Ziess Microscopy, Jena, Germany) and images were acquired using Zen Black 2012 (black edition) software (ZEISS).