Zen 2012 blue software

Fluorescence images were taken using a Zeiss LSM 700 imaging system (Carl Zeiss, Germany) equipped with LD Plan Neufluor 20× objective (NA 0.4, WD 7.9 mm). Images were analysed using ZEN 2012 Blue software (Carl Zeiss). Confocal imaging of subcellular co-localization and three-dimensional imaging of spheroids was carried out on the same system in confocal mode using solid-state laser lines 405, 488, 555 and 639 nm. Confocal images were taken with C-Apochromat 40× water immersion objective (NA 1.8, WD 0.28 mm). Analysis was carried out using ZEN 2011 Black software (Carl Zeiss).

For detection of laminin α2, 4.0 × 10⁴ CHO cells per well were seeded in 8-well glass chamber slides (Lab-Tek). Two days later, cells were washed with PBS, fixed with 4% paraformaldehyde (PFA) in phosphate buffer (PB-PFA) for 10 min, blocked with PBS containing 1% (wt/vol) BSA, and incubated with anti-laminin α2 antibody (1:500, clone 5H2; EMD Millipore), followed by Alexa Fluor 488-conjugated anti-mouse IgG (10 μg/ml; Life Technologies, Inc.). For actin staining after incubation with EPEC, CHO cells (1.0 × 10⁵ per well) were seeded in 4-well glass chamber slides (Lab-Tek). The next day, cells were incubated with or without ~2 × 10⁵ CFU of log-phase-grown EPEC for 2.5 h at 37°C. Cells were washed twice with PBS, fixed with 4% PB-PFA for 10 min, washed twice with PBS, permeabilized using 0.2% (vol/vol) Triton X-100 for 8 min, blocked with PBS containing 1% (wt/vol) BSA, and stained for actin using Alexa Fluor 488-conjugated phalloidin (1:100; Life Technologies, Inc.). The slides were mounted in SlowFade (Life Technologies, Inc.). Images were acquired using a Zeiss Axio Observer D1 microscope and Zen 2012 Blue software.

Worms were mounted on a 2% agarose pad in 0.1% sodium azide and images were taken at x40 magnification on a Zeiss Imager. M2 microscope using Zeiss Zen 2012 Blue software. It should be noted that the fluorescence intensity, gain and exposure settings remained constant for each image and the focal plane was made consistent across worms (by focusing on the grinder in the second pharyngeal bulb). Images were all 2584 × 1936 pixels in size and using ImageJ software v1.51w, the image was converted to an RGB stack. Within ImageJ, a box was drawn with one side touching the grinder. For each image, the box remained the same size (usually 1000 × 1000 pixels). The Nile Red “fluorescence” value was inferred from the Z-axis profile value for the red filter within the drawn box for each worm. Averages were taken of 20 independent animals and these were plotted with the error bars as standard error of the mean. Nile Red “fluorescence” values of the drug exposed animals were compared to the non-exposed control animals using a 2-tailed-2-sample t-test. Representative images were compiled in Adobe Photoshop 7.0.

To detect amyloid deposits, a total of 20 (2 brains of each subgroup) brains were investigated. 50 μm thick sagittal brain slices (n = 21 per brain) including sensory cortex and hippocampus were fixed on microscope slides in −20°C acetone for 20 min. The staining protocol has been described previously [43 (link)–45 (link)]. After drying at room temperature, each slice was washed twice with wash solution (PBS/Ethanol denaturised with MEK in 1:1 ratio) for 10 minutes. Then methoxy-X04 solution (10 mg methoxy-X04 powder (Tocris, Bioscience) diluted in 100 μl Dimethylsulfoxid, mixed with 450 μl of 1,2-Propandiol, 450 μl of PBS, and 50 μl 1 N NaOH; 800 μl of this stock was diluted with 200 ml of a 1:1-PBS/ethanol solution) was applied to the slices on a shaker in the dark for 30 minutes. To remove the unbonded methoxy-X04, brain slices were washed three times with wash solution and twice with distilled water for 10 minutes per step. In a final step, brain slices were preserved in fluorescence mounting medium (DAKO, Santa Clara, California, USA). Methoxy-X04 has a high binding affinity for amyloid deposits. The stained brain slices were imaged by magnification using fluorescence microscopy in tile scan mode (ZEISS Axio Imager, ApoTome.2 and Zen 2012 Blue Software, Oberkochen, Germany).

Retinal sections were viewed on a Zeiss Axio ImagerZ2 equipped with Apotome 2 and Zen 2012 blue software (Carl Zeiss, Jena, Germany). Objectives lens used were EC Plan Neofluar ×20/0.5 Ph2, EC Plan Neofluar ×40/1.3 Ph2, EC Plan Apochromat ×63/1.4 Ph3. Series of XZ optical sections (<1 μm thick) were taken at 1.0 μm steps throughout the depth of the section. Final images are presented as a maximum projection and adjusted for brightness and contrast in Adobe Photoshop CS6 (Adobe, San Jose, CA).

Astrocytes were grown on cell culture dishes with a glass bottom (Greiner; 65,000 cells/cm²). The cells were loaded with Fluo-4 A.M. (4 μm, Invitrogen) for 20 min and then incubated for a further 30 min in 37°C before measurement. Imaging was performed in normal extracellular solution (NES) containing 136 mm NaCl, 2.5 mm KCl, 10 mm HEPES, 1.3 mm MgCl₂, 10 mm glucose, and 2 mm CaCl₂, pH 7.3 (Royle et al., 2008 (link)) as described previously (Terunuma et al., 2015 (link)). In some experiments, extracellular calcium was chelated with 2 mm EGTA added into the buffer. Fluorescence images were acquired with a Zeiss Cell Observer Spinning Disk Confocal microscope using epifluorescence illumination (excitation filter bandpass 470/20 nm, emission 525/50 nm), LD LCI Plan-Apochromat 25×/0.8W objective, Zen 2012 Blue software (Carl Zeiss) and Hamamatsu ORCA-R2 camera (Hamamatsu). Images were captured at 1-s intervals for up to 2 min (in some experiments up to 4 min) in 37°C and 5% CO₂. Image data were analyzed by Zen 2012 Blue software and subsequently by OriginPro 2016 software (OriginLab). The change in intracellular free calcium concentration ([Ca²⁺]_i) is represented by relative fluorescence intensity [(F₁ – F₀)/F₀, relative unit (r.u.)] (F₀, at rest; F₁, after administration of drugs, background subtracted) in the selected cytoplasmic or nuclear parts of the cells.