Qwin image analysis software

Freshly isolated MPCs, P2-MSCs, and HUVECs were seeded at confluence in six-well plates and left to attach overnight. Cultures were then incubated for 4 h at 37 °C with 5 μg/ml AlexaFluor488®-conjugated acetylated-low density lipoprotein (Ac-LDL; Thermo Scientific) in DMEM/1% BSA. Cells were washed twice and pictures taken as already described using an inverted fluorescence microscope. Binary images were obtained by Qwin® Image Analysis Software (Leica) to quantify fluorescent areas.

On day 40 after MOG immunization, mice were anesthetized and perfused transcardially with 4% paraformaldehyde in PBS. Spinal cords were removed, fixed for 24 h in ethyl alcohol (60%), acetic acid (10%), and chloroform (30%), and embedded in paraffin. Spinal cord sections were cut at 30 mm and stained with H&E to reveal CNS inflammatory infiltrates. For immunohistochemistry, 30 mm sections were first soaked in 3% hydrogen peroxide to block endogenous peroxidase activity and then stained with anti-CD4 (clone RM4-5; Abcam), followed by the appropriate biotinylated secondary antibody (Vector Laboratories) and streptavidin-HRP (Zymed). Controls consisted of isotype-matched antibodies. For myelin staining, slides were incubated with Luxol fast blue stain (e-bioscience) for 18 h at 56°C, according to the manufacturer's protocol. Analysis was performed using QWin image analysis software (Leica). Percentage of demyelination was calculated manually measuring all areas of grey matter and the area of demyelination using the following formula, (Area of demyelination/Total area of white matter) × 100.

The proteoglycan production in chondrocytes was evaluated using Alcian blue (Muto Pure Chemicals, Tokyo, Japan) staining as previously described58 (link). Stained chondrocytes were captured by a flathead scanner, and then images were quantified via QWin image analysis software (Leica, Wetzlar, Germany).

Frozen section specimens of brain tissue were washed with 0.01 M phosphate-buffered saline and then incubated in 50% formamide/2x SSC (0.3 M NaCl, 30 mM Na citrate) in a 65°C water bath for 2 h. After washing with 2x SSC, the specimens were treated with 2 N HCl at 37°C for 30 min, followed by treatment with pH 8.5 borate buffer at room temperature for 10 min. After being washed with 0.01 M PBS, the specimens were treated with PBS containing Tween 20 (PBST) for 15 min at room temperature and then blocked for 1 h in blocking buffer (5% goat serum, 0.1% bovine serum albumin, and 0.1% Triton X-100). Thereafter, the specimens were incubated with each of the primary antibody mixtures (nestin, 1 : 200; BrdU/GFAP, 1 : 200; BrdU/MAP-2, 1 : 200; BrdU/S100B, 1 : 200; and BrdU/Galc, 1 : 200) at 4°C for 36 h. After being washed with 0.01 M PBS, the specimens were incubated with a secondary antibody solution (conjugated goat anti-mouse IgG and/or goat anti-rabbit IgG) and in the dark inside a cassette at 37°C for 2 h. The specimens were then washed with 0.01 M PBS and mounted using 50% glycerol and then were observed and photographed under a fluorescence microscope. The number of doubly positive cells in the brain tissue on the injured side in each group was counted at different time points using Leica QWin image analysis software.

We performed HUVEC migration assay by a transwell chamber with 8 μm pore size (BD Biosciences, USA). The upper chamber was plated with 1 × 10⁵ HUVECs suspended in serum-free DMEM, and the bottom chamber was added with 600 μl hAEC-CM. After 24 hours, we fixed the filters with 10% formalin, stained them with 0.1% crystal violet (Sigma, USA), and then counted the migrated cells by Leica QWin image analysis software. Complete medium was treated as a positive control medium, and serum-free high-glucose DMEM was used as a negative control medium.

At the end of the differentiation period, the cells were observed under an inverted light microscope (Leica DMIL) and pictures of the 3 fields in each well were taken. Morphometric analyses were performed using Qwin Image Analysis Software (Leica). The length of the longest neurite was measured per cell via drawing a straight line along the neurite, as previously described in [18 (link)]. Only neurites that were equal to or longer than twice the cell body diameter were included in the outgrowth analyses. Data from the 3 fields in each well were pooled and the lengths of over 150 neurites were included for statistical analysis. The neurite outgrowth experiments were performed in triplicate.