Quantification of Osteoclasts in Murine Spine

For the staining of tartrate-resistant acid phosphatase (TRAP), lumbar spine paraffin sections from two-week-old mice were incubated in a substrate solution (40 mmol/L sodium acetate, 10 mmol/L sodium tartrate, 1.6 mmol/L fast red violet, 700 mmol/L naphthol, pH 5; all chemicals Sigma-Aldrich) for 90 min and counterstained with Mayer’s hematoxylin (Sigma-Aldrich). Quantification was performed using the Osteomeasure (OsteoMetrics, Inc.) system as follows: The hypertrophic zone and underlying primary spongiosa were marked as “bone” and TRAP-positive cells were marked as “osteoclasts”. The parameter number of osteoclasts per bone perimeter (N.Oc/B.Pm) was derived, which represents the number of TRAP-positive cells per tissue perimeter (TRAP⁺ cells/T.Pm in mm⁻¹).