Tungsten knife

LR White resin embedded samples were sectioned using a Leica RM2265 microtome (Leica Biosystems, Germany). The samples were trimmed until the tissue of interest was reached. Using a tungsten knife (Leica), at a 4°angle, sections of 5 μm thickness were obtained. The sections were placed in a drop of 40% acetone on a microscope slide. The slides were placed on a hot plate at 70°C for at least 1 h, after which they were stained.

Bone samples were divided for histologic and histomorphometric evaluation. Samples were cut and fixed in 10% (w/v) formalin (Bio-Optica) for 3 days. For histomorphometric analysis, undecalcified samples were processed for plastic embedding, using a previously described protocol. Briefly, samples were dehydrated in a graded series of ethanol [50, 70 and 100% (v/v)], followed by immersion in xylol for 24 h. The undecalcified bone samples were infiltrated with a plastic embedding mixture using a three-step protocol. In each step, the samples were infiltrated for three consecutive days with daily freshly made solutions, containing 75% (v/v) of methylmethacrylate (MMA, Sigma-Aldrich) and 25% (v/v) of dibutyl phthalate (Prolabo) with increase in concentrations (0 g/mL; 0.01 g/mL and 0.025 g/mL) of benzoyl peroxide (Sigma-Aldrich). Polymerization was carried out at 37 °C for a week. The plastic blocks, containing the processed bone samples, were cut into 7 µm sections using a tungsten knife (Leica). After deplasticization, the sections were stained with toluidine blue staining. Trabecular separation (Tb.Sp) was determined using the Osteomeasure bone histomorphometry software (OsteoMetrics, OsteoMetrics, Inc.). The percentage of adipose tissue was calculated using a 15 to 11 points grid [38 (link), 39 (link)].