Leica qwin image analysis software

The formalin-fixed sciatic nerve specimens were processed for paraffin embedding before being cut into 4-μm sections and then stained with hematoxylin–eosin (HE) staining and toluidine blue for light microscopic examination (Culling 2013 ). The extent of sciatic nerve fiber degeneration, Schwann cell loss, and inflammatory cells infiltration was used for grading the severity of the pathologic changes in the HE-stained sections. A 4-point scoring scale was used with 0, 1, 2, and 3 indicating no (0%), mild (1–25%), moderate (26–50%), and severe (> 50%) pathological changes, respectively (Ibrahim et al. 2020 (link)). Furthermore, the number of myelinated nerve fibers for each group was quantified by Leica QWin image analysis software (version 3; Leica Microsystems Ltd, Heerbrugg, Switzerland) using 5 random non-overlapping microscopic fields for each toluidine blue-stained section.

Paraffin-embedded sciatic nerves sections (4-μm thick) were used to evaluate MMP-2 and MMP-9 expression. Retrieved specimens were treated for 30 min at room temperature with 3% hydrogen peroxide/methanol, then washed with phosphate-buffered saline. Sections were treated with 10% goat blocking serum for 1 h at room temperature. Later, specimens were incubated with MMP-2 or MMP-9 rabbit monoclonal antibodies (1:100 dilution; Catalog No: MA5-13590 and MA5-14228, respectively, Thermo Fisher Scientific, Hanover, IL, USA) overnight at room temperature. After washing, sections were incubated with biotinylated secondary antibody (Dako, Glostrup, Denmark) and then with horseradish peroxidase-conjugated streptavidin for 60 min each at room temperature. Three additional washes were performed, then the reaction was visualized by 3,3′-diaminobenzidine tetrahydrochloride (DAB Substrate Kit, Vector Laboratories Inc., Burlingame, CA, USA). Slides were counterstained with hematoxylin, dehydrated, mounted, and examined by a light microscope. The area percentage of immunopositive cells to the total area of the microscopic field was analyzed by Leica QWin image analysis software (version 3; Leica Microsystems Ltd, Heerbrugg, Switzerland) at ×400 magnification. The analyses were conducted using 5 non-overlapping microscopic fields that have been randomly chosen from each section.

Bone slices (Immunodiagnostic Systems Limited, Boldon, UK) were put into 24-well culture plates, and nonadherent BMMs were plated on top of them. The cells were incubated with M-CSF (20 ng/mL) for 3 days before they were stimulated with RANKL (100 ng/mL) and various doses of triptolide. The culture medium was exchanged for fresh medium every 2 days in order to maintain a constant concentration of M-CSF and RANKL. After culturing the cells for 12 days at 37°C in a 5% CO₂ incubator, the cells were removed from the Bone slices by sonication in the presence of 0.25 M NH₄OH, and then the Bone slices were gradient dehydrated in 40%, 75%, 95%, and 100% ethanol and stained with 1% toluidine blue (Sigma-Aldrich, St. Louis, MO, USA) for 5 minutes. Finally, the areas of the bone resorption pits were calculated using the Leica Qwin image analysis software (Leica Microsystem, Germany).