Comprehensive Bone Tissue Analysis Protocol

After micro-CT analysis, femurs were included in methyl methacrylate (MMA) at 4°C. Serial 5μm sections were cut in three distinct levels, eight sections per level, with a 50μm interval between levels. Relative osteoid surface (Osteoid / Bone surfaces = OS/BS *100%) and number of osteoblast per bone surface N.Ob/BS (mm^-1) were determined in 2 sections per level in each animal by staining with toluidine blue (1%) as described previously [19 (link)]. The Axioimager Z2 microscope (Zeiss, Germany) was used for osteoid and bone surfaces identification and each area was measured with Axiovision software (Zeiss, Germany) at 200x magnification. Bone and osteoid surfaces were measured in the distal metaphysis of the femur. To determine the osteoblasts number (N.Ob) in OS a magnification of 400x was used (Axioimager Z2 microscope, Zeiss, Germany). Osteoclasts were stained with naphthol AS-TR (3- hydroxy-2-naphthoic acid 4-chloro-2-methylanilide) phosphate for tartrate-resistant acid phosphatase (TRAP) detection and counterstained with Toluidine blue (0.5%), [20 (link)]. Osteoclasts were counted and expressed relatively to bone surface as Oc.S/B.Ar (mm²). To evaluate total iron accumulation in femur trabecular bone, sections were stained with Perl’s solution ((2% Ferrocyanide potassium) 1:1 (2% HCl (37%)) and counterstained with Nuclear Fast Red (1%).