2.2  Immunohistochemical staining for FoxP3, CD4 and CD3
Bone marrow specimens from posterior iliac crest were obtained in all cases. Immunohistochemical staining for FoxP3, CD4 and CD3 was separately performed using Ventana Benchmark GX Immuno-AutoStainer (Roche) according to the manufacturer’s instructions. The primary anti-human rabbit monoclonal antibody, incluing anti-FoxP3 (dilution 1:500; Abcam), anti-CD4 (dilution 1:100; Abcam) and anti-CD3 (dilution 1:100; Abcam), were revealed with an indirect biotin streptavidin system (HRP/DAB Detection ICH kits, Abcam). Cells of FoxP3+presented brown chromogen on the nucleus, while CD4+and CD3+ cells showed cytoplasmic brown granules.
All slides were reviewed independently by two skilled pathologists. The positive cells were counted in 5 visual fields near endosteum of trabecular bone under high-power magnification (×600). The mean value of FoxP3+/CD4+ in each case represented the local immune tolerance status mediated by Tregs.