Xc10 camera

At 24 h after seeding the cells in 96-well plates with blacked walls (Corning, Amsterdam, The Netherlands), the tested compounds were applied at various concentrations. After 48 h of treatment, the cells were gently washed with PBS, fixed with 4% paraformaldehyde (Avantor Performance Materials, Gliwice, Poland) in PBS (4 °C, 10 min), permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, Poznań, Poland) in PBS (4 °C, 15 min), labeled with DyLight™ 554 Phalloidin (#13054, Cell Signaling Technology, Warsaw, Poland) for 10 min and DAPI (Thermo Fisher Scientific, Warsaw, Poland) for 5 min, visualized using an Olympus IX81 fluorescence microscope equipped with a XC10 camera and analyzed with CellSens Dimension software (all Olympus Polska, Warsaw, Poland).

For light microscopy (LM), specimens were washed six times in phosphate buffered saline (0.1 mol l^− 1) over a period of 24 h, with one step in a low vacuum to ensure that the tissue was free of air bubbles. Samples were then dehydrated through a graded series of ethanol (30–96%) with the final two steps in a low vacuum. The samples were embedded in hydroxyethyl methacrylate (Historesin; Leica Microsystems, Wetzlar, Germany). Histological sections were cut at 2 μm thickness using an AO Spencer No. 820 rotary microtome or a Microm HM 340 E electronic rotary microtome (Thermo Fisher Scientific Inc., Waltham, MA, USA). The sections were stained using Rüdeberg staining solution (0.1% methylene blue, 0.1% thionin and 0.1 mol l^− 1 Na₂HPO⁴ in distilled water; [26 ]). For histological analysis, we used either an (i) Olympus BX51TF microscope (Olympus, Hamburg, Germany) equipped with a microscope camera (UCMOS camera, ToupTek Photonics, Hangzhou, P. R. China) and image capturing software ToupView (ToupTek Photonics, Hangzhou, P. R. China), or (ii) an Olympus BX61VS scanning microscope equipped with an Olympus XC10 camera and VS-AWS FL 2.8 scanning and capturing software, or (iii) a Zeiss Axiophot equipped with an AxioCam ERc5s camera and Zen 2.3 blue edition (Zeiss 2011).

The tendon specimens were stained with hematoxylin and eosin (H&E). Photomicrographs of the tissue were captured through an Olympus IX71 light microscope and an Olympus XC10 camera (Japan). Four sections of each sample were selected and evaluated by two blinded observers to assess the tendon morphology according to a modified semi-quantitative grading score from 0 to 3. The score analyzed the fiber arrangement, fiber structure, nuclear roundness, cell density, infiltration of inflammatory cells and fibroblasts, and neovascularization [18 (link)–20 (link)]. According to this grading system, a perfectly normal tendon scored 0 and mild and moderate prevalence scored 1 and 2, whereas a score of 3 was assigned to a severely abnormal tendon.

Wild type adult zebrafish (AB line) reared at 28 °C with
the light cycle of 14 h light/10 h dark were
group-mated. Spawned eggs were staged according to Kimmel et al.53 (link). Embryos at 24 h postfertilization were pretreated
with O-(Carboxymethyl) hydroxylamine hemihydrochloride (AOAA, Sigma, Cat.
#C13408) 100 μM for 2 h in E3 egg water at
28.5 °C incubator, followed by three time
(5 min each) washes with E3 egg water. After washes, embryos were
transferred to L₁ solution with two concentrations of
5 μM or 25 μM for
30 min in E3 egg water at room temperature, again with three time
(5 min each) washes with E3 egg water afterwards. The embryos were
embedded alive in the 2.5% methyl cellulose, and fluorescence signals were
visualized under the Olympus SZX16 stereo microscope equipped with the
excitation filter GFP-A illuminated using a mercury lamp (Olympus, U-RFL-T).
Images were captured using Olympus XC10 camera. All zebrafish husbandry and
animal care were carried out in accordance with guidelines from the Korea
Research Institute of Bioscience and Biotechnology (KRIBB) and all experimental
protocols were approved by KRIBB-IACUC (approval number: KRIBB-AEC-16036).

Being explanted tendons very long, before fixation and inclusion, each sample was divided for the entire width at its middle thirds into a proximal and a distal portion, representing the areas of transition between the native tendons and the grafts. Then, the specimens were fixed in 10% neutral buffered formalin for 24 h and dehydrated in alcohol scale before embedding in paraffin and cutting into 4 μm longitudinal sections. The slides were stained with Alcian Blue (AB). Photomicrographs of the tissue were captured through an Olympus IX71 light microscope and an Olympus XC10 camera (Japan). Three independent, blinded examiners assessed the tendon histopathology according to a modified semiquantitative grading score, as proposed by others [20 (link), 21 (link)]. In particular, the following parameters were evaluated: organization of the extracellular matrix (0-2), cellularity (0-2), cell alignment (0-2) and distribution (0-1), the morphology of the cell nuclei (0-2), organization of repair tissue of the tendon callus (0-2), transition from defect to normal tissue (0-2), vascularization (0-1), degenerative changes (0-3), inflammation (score 0-1), and proteoglycan content (0-1). The maximum score (19) represented the best outcome in terms of tendon tissue integrity, and the minimum value (0) represented the worst condition.

Paraffin embedded sections were deparaffinised in histolene, rehydrated in an ethanol gradient (100, 70 and 30%) and then immersed in a PicroSirius staining solution (0.1% w/v of Sirius Red F3BA in a saturated aqueous solution of picric acid) for 1 h at room temperature. After the incubation, the slides were washed three times in a solution of 0.5% glacial acetic acid. Sections were rapidly dehydrated using 100% ethanol, immersed in histolene and finally mounted using DPX mounting medium (Sigma-Aldrich, 100579). Mounted slides were dried overnight, and whole tissue section images were captured on the DotSlide system at × 10 magnification using an Olympus XC10 camera located in the Monash Micro Imaging facility (MMI) (Clayton, Australia). The area of positive staining was quantified as previously described^{14 (link)}. Briefly, ImageJ was used to quantify the area of positive stained ovarian tissue above a threshold that was set based on the staining in the oldest animal. The same threshold value was used for all images analysed.