Polycut e microtome

Bones were fixed overnight at 4°C in 70% ethanol solution and dehydrated in a graded ethanol series. Undecalcified samples were embedded in methyl methacrylate (Merck, Rahway, NJ). Serial sections, 4 μm thick, were cut on a microtome (Polycut E microtome, Leica, Wetzlar, Germany). Series of consecutive sections representative of micro-CT images were stained respectively with toluidine blue (pH 3.8), von Kossa reagent (5% silver nitrate solution, Sigma-Aldrich, St Louis, MO), Masson’s trichrome staining (Sigma-Aldrich, St Louis, MO) and safranin O staining (Weigert’s iron hematoxylin, Sigma-Aldrich, St Louis, MO; Fast Green, Prolabo, Paris, France; and Safranin O solution, Sigma-Aldrich, St Louis, MO).

Human cartilage samples were fixed in 4% paraformaldehyde for 72 h before paraffin embedding. Transverse sections (4 µm thick) were cut parallel to the rib axis by use of a Polycut E microtome (Leica, Wetzlar, Germany), then mounted on slides. For labeling, slides were deparaffinized and unmasked by use of citrate buffer (pH 6) at 95°C, then blocked in a solution of TBS-TC [0.745 g/l Tris-HCl, 9 g/l NaCl, 0.02% Tween 20, 0.6 g/l caseine (Sigma), pH 7.4]. Human CD13 was detected with monoclonal rabbit antibody (AB108310; Abcam). Rabbit IgG (DAKO) was a negative control. Secondary antibody and peroxidase were added by use of the horseradish-perioxidase-conjugated LSAB+ system (DAKO). Visualization involved the DAB kit (VECTOR). Image-Pro express was used for image capture.

After sacrifice, bones were fixed overnight at 4°C in 70% ethanol solution and dehydrated in a graded ethanol series. Undecalcified samples were embedded in methyl methacrylate (Merck, Rahway, NJ). Serial sections, 5 μm thick, were cut on a microtome (Polycut E microtome, Leica, Wetzlar, Germany). Series of consecutive sections were stained with Von Kossa (5% silver nitrate solution; Sigma-Aldrich), counterstained with toluidine blue (pH 3.8), and stained with Safranin O Lillie’s Trichrome (Sigma-Aldrich).