Cell b software

Skeletal muscle immersed in the formalin solution was dehydrated and embedded in paraffin wax. Three slices with 3-μm thickness of each sample were cut and stained with hematoxylin and eosin to observe the morphology of muscle. The stained slices were photographed using an Olympus CX41 microscope at 40 × magnification. The Olympus Cell B software (Olympus) was used to measure myofiber characteristics. Muscle fibers from 5 random fields of each sample were chosen to measure and calculate the fiber diameter and cross-sectional area. The total fiber number of each field was counted and was converted to fiber density by dividing the area of the field. Fiber density was expressed as total fiber number per mm² muscle.

Liver tissue samples were withdrawn for further histological analysis. For the histopathologic investigation of the liver, the harvested samples were fixed in neutral 10% buffered formalin and were subsequently embedded in paraffin, sectioned at 4 μm thicknesses (Leica rotary microtome, RM2125, Nussloch, Germany) and stained with hematoxylin and eosin (H&E) and PAS methods (Periodic acid–Schiff staining). Histologic examination was performed with the aid of an Olympus BX51 microscope connected to a digital camera (Olympus DP-25, Tokyo, Japan). The microphotographs were acquired using an Olympus system for image acquisition and analysis (Olympus Cell B software, Tokyo, Japan).

Acid-free protocols were adapted [65 (link)] to perform Alcian blue (8 GX Sigma-Aldrich, Diegem, Belgium) staining of cartilage structures and Alizarin red S (Sigma-Aldrich, Diegem, Belgium) staining of calcified structures. At 6, 9 or 10dpf, the larvae were fixed in 4% PFA for 2h at room temperature and rinsed several times with PBST.
Cartilage was stained overnight in 10 mM MgCl₂, 80% EtOH and 0.04% Alcian blue. The larvae were washed in different concentrations of ethanol (80%, 50%, 25%) to remove excess staining. Pigmentation was bleached in a H₂O₂ solution (H₂O₂ 3%, KOH 0.5%) and finally the larvae were rinsed 3 times in a solution of 25% glycerol / 0.1% KOH and 50% glycerol, 0.1% KOH and finally stored in this solution at 4°C.
During acid-free bone structure staining with Alizarin red, bleaching was performed immediately after fixation, before the staining. After the bleaching, long rinses (at least 20min each) in a 25% glycerol, 0.1% KOH solution are necessary to prevent the fading of the staining. The larvae are stained in a 0.05% Alizarin red Solution in water for 30min in the dark on low agitation, rinsed in a 50% glycerol, 0.1% KOH solution to remove excess staining and kept at 4°C in the same solution.
Images of stained larvae (n = 20–30 larvae) were obtained on a binocular (Olympus, cell B software).

Phytoplankton samples, collected in each sampling site, were visualized under an optic microscope (Leica DMLB, Germany) and isolated through the micromanipulation technique using a Pasteur pipette. Both colonial and filamentous cyanobacteria strains belonging to M. aeruginosa, R. raciborskii and P. agardhii were isolated and transferred into culture tubes supplemented of 5 mL of Z8 culture medium to promote cyanobacterial growth [28 ]. Isolated microorganisms were maintained in culture under 25 °C, with a photoperiod of 14 h:10 h light-dark and 25 μEm2s of light intensity without aeration. Cultures were transferred to 50 mL culture flasks with filter caps (Orange Scientific, Braine-l’Alleud, Belgium) containing Z8 culture medium and maintained in the same growth conditions. Cultures were visualized in an Olympus BX41 optic microscope coupled with an Olympus DP72 photograph machine and using Olympus Cell^B software for image acquisition.

Live and stained embryos were photographed on an Olympus BX51 compound microscope equipped with DIC optics, using a Camedia C-3030ZOOM camera and CELL-B software (Olympus). High-speed video microscopy was undertaken and time-to-colour merges of movies were made as described (Stooke-Vaughan et al., 2012 (link)). Fluorescent samples were imaged on a laser-scanning confocal microscope (Leica SP1 or Nikon A1) or a spinning disc confocal system (PerkinElmer Ultraview Vox with an Olympus IX81 microscope). Images were assembled using Adobe Photoshop and Fiji (ImageJ) (Schindelin et al., 2012 (link)).

To assess in vivo tumor formation and angiogenesis, the CAM assay was used, as previously described [33 (link)], with some modifications. Briefly, fertilized chicken eggs (Pinto Bar, n = 5 for each experiment) were incubated at 37°C;C and 70% humidity. On day 3 of development, after puncturing the air chamber, a hole in a specific region of the eggshell was performed and eggs were sealed with tape and returned to the incubator. On day 10, a small plastic ring was placed on the CAM and 5 × 10⁶ PCa cells (PC3 or LNCaP, control and sh-SMYD3), ressuspended in 20 μL of RPMI or RPMI/F12 medium, were injected in the ring over the CAM. On day 14, the tumor formed was photographed in ovo using a stereomicroscope (Olympus S2x16, Olympus, Tokyo, Japan) and, on day 16, chicks were sacrificed at −80°C; C for 10 minutes. The CAM and tumors were fixed with 4% paraformaldehyde and photographed ex ovo. Samples (n = 10 for each experimental condition) were paraffin-embedded, sectioned, and stained with hematoxylin and eosin (HE) for histological analysis. The total area occupied by tumors was measured using the Cell B software (Olympus) and linear vessel density was assessed by calculating the ratio between the number of vessels and the total length of the membranes.