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Commensal microbiota in vivo regulation of osteoclastogenesis. 12 week-old male SPF & GF mice were euthanized; (a–g) femurs harvested for histomorphometric analyses (n = 4/gp), and (h–j) bone marrow and calvaria were harvested for gene expression analysis (n = 4/gp). (a–g) Histomorphometric analyses of osteoclast cellular endpoints and resorbed bone were performed in the trabecular bone secondary spongiosa of tartrate-resistant acid phosphatase (TRAP) stained distal femur sections; TRAP + cell lining bone with ≥ 3 nuclei designated an osteoclast. (a) Representative images of TRAP-stained secondary spongiosa (400×). (b) N.Oc/B.Pm = osteoclast number per bone perimeter. (c) Oc.Ar/Oc = average osteoclast area. (d) Oc.Pm/B.Pm = osteoclast perimeter per bone perimeter. (e) E.Pm/B.Pm = eroded perimeter per bone perimeter. (f) Oc + E.Pm/B.Pm = osteoclast-positive eroded perimeter per bone perimeter. (g) Oc-E.Pm/B.Pm = osteoclast-negative eroded perimeter per bone perimeter. (h–j) qRT-PCR analysis in bone marrow and calvaria to assess alterations in the RANKL/OPG Axis. (h) Tnfsf11(Rankl) mRNA in bone marrow and calvaria. (i) Tnfrsf11b(Opg) mRNA in bone marrow and calvaria. (j) Tnfsf11(Rankl):Tnfrsf11b(Opg) ratio in bone marrow and calvaria. Relative quantification of mRNA was performed via the comparative CT method (ΔΔCT); Gapdh was utilized as an internal control gene; data expressed as fold difference relative to SPF. Data reported as mean ± SEM. *p < 0.05 vs. SPF; **p < 0.01 vs SPF; ***p < 0.001 vs SPF.