H550s photo imaging system

Histologic analyses (knee joint) were carried out to assess the tissue morphology with particular attention toward signs of inflammation, bone necrosis and osteomyelitis. After decalcification [0.3 M EDTA, 28 days] (bone), dehydration (ethanol, xylene, and paraffin), and paraffin-embedding (all samples), samples were sectioned (4 μm) and stained with hematoxylin and eosin (H&E). All the slices were observed and photographed by H550S Photo Imaging System (Nikon, Japan), and assessed by an experienced observer blinded to treatment.

The chondrocytes cultivated were washed three times using ice-cold PBS, fixed in 4% formaldehyde for 15 min, and blocked for 1 h with 3% BSA. The cells were then incubated overnight at 4 °C with primary antibodies, including rabbit anti-Col2a1 (1:200 dilution), anti-MMP13 (1:200 dilution), and anti-p65 (1:200 dilution). The cells were washed and incubated with goat anti-rabbit secondary antibody (1:200 dilution) for 1 h at room temperature. The nuclei were stained using DAPI at 1:500 dilution for 5 min. The slides were washed and fluorescence images were captured using the H550S Photo Imaging System (Nikon, Tokyo, Japan). The staining intensity was determined by measuring the mean optical density in 10 different fields for each sample.