Evaluation of the osteoclastic phenotype by staining for TRAP and evaluation of its gene expression. (A) Fast Green staining of trabecular bone (green) and TRAP staining (red, see arrows) of the knee joints of F8^−/− mice that were not subjected to needle puncture (unpunctured), or were punctured and were left untreated or treated with etanercept, or were punctured with additional genetic inactivation of TNF-α or iRhom2 (left panels: original magnification ×40; right panels: original magnification ×200). The micrographs shown are representative for the average sample of each treatment group. (B) Quantification of the osteoclast surface compared with the total bone surface (Oc. S/BS) in unpunctured versus punctured joints (11% ± 1% and 26% ± 4%, respectively, for F8^−/− mice [n = 6]; 10% ± 1% and 10% ± 1%, respectively, for etanercept-treated F8^−/− mice [n = 7], 11.6% ± 0.7% and 11.5% ± 1%, respectively, for F8^−/−/TNF-α^−/− mice [n = 7]; 8.4% ± 1% and 9.8% ± 1%, respectively, for F8^−/−/ iRhom2^−/− mice [n = 9]). (C) qPCR analysis of TRAP gene expression (fold change) compared with the unpunctured joint (3.3-fold for F8^−/− mice [n = 5]; 0.8-fold for etanercept-treated F8^−/− mice [n = 3]; 1.4-fold for F8^−/−/TNF-α^−/− mice [n = 4]; and 1.7-fold for F8^−/−/iRhom2^−/− mice [n = 4]). *P < .05 vs unpunctured untreated, **P ≤ .05 vs punctured untreated.