Histological Analysis of Inflammatory Joint Changes

At day 10, both forepaws and the entire right hind limb, including the paw, ankle, and knee were surgically removed and fixed immediately in 10% buffered formalin (Biochemical Sciences). The preparation of knee joint samples and histological analysis using toluidine blue (T blue) dye were performed as previously described (14 (link), 15 (link)). All sections were read by a trained observer who was also blinded to the treatment and to the disease activity score of each mouse. The joint sections from mice were scored for the changes in inflammation, pannus, cartilage damage, and bone damage on a scale of 0–5. C3 deposition in the joints (synovium and cartilage) was localized with polyclonal goat anti-mouse C3 antisera (ICN Pharmaceuticals) and scored as previously described (13 (link)). Macrophages infiltration in the knee joints were localized with rat anti-mouse F4/80 Ab (Bio-Rad) and scored using a scale from 0 to 4.