Immunohistochemistry

Immunohistochemical staining was performed using a Ventana Benchmark® XT automated slide preparation system (XTUltraviewDABv3, Ventana, Tucson, AZ, USA). 4-μm thick sections were incubated with primary antibody (Table 2). After washing in phosphate-buffered saline (PBS), sections were incubated with streptavidin/horseradish peroxidase (Biolegend, San Diego, CA, USA). Sections were immersed in 3% H₂O₂ to quench endogenous peroxidase activity. Staining was visualized in brown color using a diaminobenzidine tetrahydrochloride chromogen substrate (DAB, SK-4105 Vector Laboratories, Burlingame, CA, USA). Sections were counterstained with hematoxylin. Slides were scored by two pathologists blinded to clinical history and the primary antibody used. The number of positive cells was evaluated for each assay from five randomly selected areas (200 HPF), except for vimentin. Vimentin expression was semi-quantitatively evaluated (0 = vimentin negative; + few positive cells; ++ less than the half positive cells; +++ more than half positive cells).

RANK/RANKL/OPG triad and NFATc1 expression by cherubism-granuloma cells

Expression of each protein was evaluated for MGC and stromal cells. For NFATc1, numbers of nuclear and cytoplasmic positive cells were evaluated. For each assay five randomly selected areas were evaluated (200× high-power field)

For immunohistochemistry staining, 4-μm thick sections were first deparaffinized with xylene for 30 min, post-fixed with 90% ethanol for 10 min and then washed in distilled water for 5 min. For antigen retrieval, the sections were incubated in a 78 °C water bath for 30 min in pH 6 or pH 9 buffer, cooled at room temperature for 20 min, washed in PBS for 10 min and finally incubated with reagents from an avidin/biotin kit (Vector Laboratories, Perterborough, UK). Endogenous perodixase activity was blocked by incubating the sections with 3% H₂O₂ followed by a wash in PBS. Sections were blocked in a 5% normal human serum for 30 min before incubation with the primary antibodies or isotype control for 60 min at room temperature. After a PBS wash, the sections were then incubated with the secondary antibody for 30 min, then with streptavidin/horseradish peroxidase for 30 min (Biolegend, San Diego, CA, USA). Staining was visualized as a brown color by using a diaminobenzidine tetrahydrochloride chromogen substrate (Vector Laboratories, Perterborough, UK). Slides were scored by two pathologists blinded to clinical history and the primary antibody used. The signal for each antibody was evaluated for MGCs and stromal cells. For NFATc1, the numbers of nuclear and cytoplasmic positive cells were evaluated.