Zen 2012 blue edition software

Protoplasts were prepared using the Tape-Arabidopsis Sandwich procedure from leaves of transgenic plants (T2 generation) expressing AtVCCN-GFP40 (link). Protoplasts were mounted in W1 solution (0.5 M mannitol, 20 mM KCl, 4 mM MES-KOH, pH 5.7) and observed using a LSM 700 inverted Axio Observer Z1 confocal laser scanning microscope (Carl Zeiss Microscopy, Jena, Germany) equipped with a LD C-Apochromat water immersion objective lens (× 40/1.1 NA). GFP and chlorophyll fluorescence were both excited at 488 nm and emitted signals were collected above 640 nm for chlorophyll fluorescence (long-pass filter) and below 548 nm for GFP fluorescence (variable secondary dichroic beamsplitter). Images were analysed with Zen 2012 blue edition software (Carl Zeiss Microscopy).

The effects of the different inhibitor treatments on osteoclast differentiation on bovine bone slices were studied by fluorescent staining of nuclei and F-actin rings. First, osteoclasts were fixed with 4% paraformaldehyde, permeabilized with 0.01% Tween-20 in PBS on ice for 5 min, and non-specific binding was blocked with 1% BSA in PBS for 30 min. Cells were stained with STAR635 phalloidin (Abberior GmbH, Gottingen, Germany; catalogue number 2-0205-002-5) in 1:100 dilution to visualize F-actin and with Hoechst 33342 at a 1:10000 dilution in 1% BSA in PBS to visualize the nuclei, both for 1 h at room temperature. Glass coverslips were embedded with 30% glycerol in PBS and imaged with Carl Zeiss Axioimager microscope and Carl Zeiss Zen 2012 (blue edition) software.

To investigate if cell membranes of non-culturable bacteria remained intact after exposure to the respective antimicrobial treatment, we used the LIVE/DEAD^TMBacLight^TM bacterial viability kit (Molecular Probes, Eugene, OR, United States). Therefore, 100 μl of the bacterial sample was removed from the respective wells, centrifuged at 8,000 ×g for 5 min, washed with PBS twice, and diluted 1:100. Then 1.5 μl of SYTO 9 dye and 1.5 μl of propidium iodide were added to the sample, followed by vortexing and 15 min incubation in the dark. After incubation, cells were trapped with a 0.2-μm polycarbonate membrane filter (Sterlitech, Kent, WA, United States), which were subsequently placed between a slide and a coverslip and used for fluorescence microscopy. Viable cells with intact membranes will appear green by fluorescent SYTO 9, which permeates intact or damaged plasma membrane, while propidium iodide can only permeate damaged and disrupted plasma membranes and competes with SYTO 9 for DNA-binding sites. Therefore, cells with intact membranes show green fluorescence while cells with damaged membranes appear with red fluorescence. Those were considered to be dead. Pictures were taken with the Zeiss Observer Z1 inverted widefield microscope and ZEN 2012 (blue edition) software (Oberkochen, Germany).

For immunohistochemistry (IHC) evaluation, bovine ovaries were selected as described above. Entire ovaries were then fixed in 10% formaldehyde solution for 24 h, rinsed, and dehydrated in alcohol until they were embedded in paraffin. Serial sections were prepared (at a thickness of 3 µm), followed by deparaffinization, rehydration, sodium citrate heat-mediated antigen retrieval, peroxidase block, and protein blocking (10% goat for 30 min), and then slides were probed with primary antibody against total and phosphorylated forms of YAP (Table 1) overnight at 4 °C. Protein detection was then performed with the Vectastain Elite ABC HRP Kit (VECTPK6101, Vector Laboratories, Burlingame, CA, USA) and stained with the DAB substrate kit (VECTSK4100, Vector Laboratories). Slides were then counterstained with hematoxylin and dehydrated with graded alcohols prior to mounting. Negative controls were included in the IHC analysis and consisted of slides for which the primary antibodies (for both total and phosphorylated YAP) were omitted. The results confirmed the specificity of our second antibody (not shown). Photomicrographs were taken using a Carl Zeiss Axio Imager M1 microscope (Carl Zeiss, Toronto, ON, Canada) at ×1000 magnification and using the Zen 2012 blue edition software (Carl Zeiss).

Images were acquired on an LSM 900 confocal microscope equipped with an Airyscan 2 detector and Zen 2012 (blue edition) software (Carl Zeiss Microscopy, Göttingen, Germany). For morphometric evaluation of MOG labeling, images of 3–4 individual lobules within the cerebellar cortex of each OSC, and from two OSCs per animal, were evaluated, after background subtraction, by calculating the percentage of immunopositive areas in frames of 350 × 350 μm using ImageJ Software (Fiji, U. S. National Institutes of Health, Bethesda, Maryland). The numbers of IBA-1 and ARG-1 immunopositive cells within the white matter and the internal granular cell layer of a cerebellar lobule were quantified in 3–4 lobules per OSC and from two OSCs per animal, using the bioimaging analysis platform QuPath (Bankhead et al., 2017 (link)), in which cells were segmented and subsequently classified. The cell segmentation model was trained under visual control on a set of five images per condition using the deep learning-based StarDist library (Schmidt et al., 2018 ).
Immunopositive cells in OPC cultures were counted at 20x magnification using an Olympus BX41 fluorescence microscope and cellSens software (Olympus, Tokyo, Japan).

At 21 and 35 days of age, two birds from each replicate were randomly dissected to collect small intestinal samples. The middle of jejunum samples was sectioned horizontally in 2 cm lengths [14 ]. All samples were fixed in 10% formalin. After fixation, the samples were dehydrated by increasing the concentration of alcohol and embedded in paraffin. Embedded tissue samples were sectioned at a 5 mm thickness using an auto-microtome (Leica RM 2155, Leica Microsystems GmbH, Nussloch, Germany). The slides were stained with hematoxylin and eosin. The villus height and crypt depth were determined in cross-section using Axiolab inverted microscope (Carl Zeiss, Hamburg, Germany) equipped with an HBO 50 camera to capture images and subsequently analyzed using Zen 2012 (blue edition) software (Carl Zeiss Microscopy GmbH, Hamburg, Germany). The measurements were examined on 10 villi and 10 crypts for each segment. The villus height-to-crypt depth ratios were then calculated.