Vs120 s6

At the end of the experiment, mice were over-anesthetized (pentobarbital 1 gr/kg, I.P) and perfused transcardially with 2.5% paraformaldehyde. The brain was removed and postfixed for 24 h in the perfusion solution. Brains were then immersed in PBS solution with additional 30% sucrose for 24 h and then cut in a freezing microtome (80 μm thick, SM 2000R; Leica, Heidelberg, Germany).
Brain slices were mounted on slides and scanned using ZEN software (Zeiss) by a fluorescent microscope (VS120-S6, Olympus). Images were exported and processed using ImageJ software.

The fresh samples were fixed in 4% paraformaldehyde for 48 h followed with decalcification in 10% EDTA-2Na for 3 weeks, and then paraffin-embedded for further routine histological preparation. Three micrometer-thick sections were deparaffnized in xylene and rehydrated in graded alcohols and distilled water prior to H.E. and Safranin O –Fast green stainings as previously described [18 (link), 19 (link)]. The articular cartilage thickness of each femur condyle (from superficial zone to tidemark) was measured using Image-Pro Plus 6.0 software. According to OARSI scoring system established for grading OA changes [25 (link)], and semi-quantitative histopathological grading was performed using VS120-S6 (Olympus, Japan) by two different blinded pathologists, for a maximum possible score of 6. Grade 0 represents normal articular cartilage and increasing grade indicates a more biologically cartilage degeneration.

H&E, IHC, and immunolabeling were analyzed using a slide scanner (VS120 S6, Olympus) equipped with a XM10 monochrome camera (Olympus) and a Pike F‐505C color camera (Allied Vision). Image scans were obtained and further processed using the following Programs: VS‐ASW 2.9, OlyVia (Olympus) and Adobe Photoshop CS6 (Adobe). For some IF analyzes, images were taken using a LSM Meta 510 confocal microscope (Zeiss). It was ensured that identical exposure times and gray value settings were used for the samples to be compared. For automated image quantification, a script was developed using Fiji software (Schindelin et al, 2012 (link)). In brief, a tissue mask was interactively created corresponding to the tissue region of interest. Particle analysis was done after applying a fix threshold to allow a comparative analysis. For enhanced particle separation, a watershed algorithm was used. Particle analysis revealed the parameters particle count, area, and area fraction of the tissue region of interest. For the quantification of Ki67, positive cells in the interfollicular epidermis (IFE), positive cells of the basal, and suprabasal layers of the IFE were counted and set in relation to the total cell number.