Labophot 2a microscope

After euthanasia, left femurs were dissected, fixed in 70% ethanol, dehydrated, embedded in methyl methacrylate, and sectioned longitudinally with a Policut S microtome (Reichert-Jung, Heidelberg, Germany) in 5 µm-thick sections. Samples were stained with 0.1% toluidine blue (pH 6.4) for histomorphometric analysis which was performed using semiautomatic method with a Labophot-2A microscope (Nikon®), and software Osteomeasure (Osteometrics, Inc, Atlanta, EUA). These histomorphometric parameters are suggested by the American Society of Bone and Mineral Research histomorphometry nomenclature committee^{52 (link)} as follow: bone volume represented as a percentage of tissue volume (BV/TV, %); trabecular thickness (Tb.Th, µm); trabecular separation (Tb.Sp, µm); trabecular number (Tb.N, /mm); osteoid volume as a percentage of bone volume (OV/BV, %); osteoid surface as a percentage of bone surface (OS/BS, %); osteoblast surface as a percentage of bone surface (Ob.S/BS, %); osteoid thickness (O.Th, µm); osteoclast surface as a percentage of bone surface (Oc.S/BS, %); and eroded surface as a percentage of bone surface (ES/BS, %).

The Alizarin red staining (Sigma-Aldrich, Dorset, UK) was used to stain calcium at days 14 and 21. Upon formalin-fixation (Genta Medical, York, UK), the samples were incubated with 40 mM alizarin red stain for 20 min for 2D and 30 min for 3D. 3D samples require more ddH₂O washes (minimum 5) than 2D samples. Images were captured via Nikon brightfield reflected light microscopy using a Nikon ‘Labophot’ 2A microscope, with 100 W epi-illumination and metallurgical objectives.