Quantification of FGF23 Expression in Tibial Bone

After dissection, the condyles at the proximal end of the tibiae were sawn off with a diamond saw and the distal end was cut off with scissors. The tibiae were immediately fixed for 24 hours at 4°C in 4% paraformaldehyde in phosphate buffered saline (PBS). After fixation the tibiae were decalcified in 10% EDTA and 0.5% paraformaldehyde in PBS at 4°C for 4 weeks. Proper decalcification was verified by x-ray photography. The tibiae were then dehydrated and embedded in paraffin and cut into 5 μm thick sections. Sections were rehydrated and endogenous peroxidase was quenched with 3% H₂O₂ in 40% methanol/PBS. Antigen retrieval was performed through incubation with 0.5% trypsin + 0.1% CaCl2 for 15 minutes at 37°C. After the blocking of the non-specific binding sites with blocking solution (Invitrogen, Waltham, MA, USA) for 1 hour, the sections were incubated overnight at 4°C with 1/1000 anti-intact FGF23 antibody (Santa Cruz Biotechnology Inc, Dallas, TX, USA). The sections were incubated with 1/100 biotinylated second antibody (Dako, Agilent Technologies, Inc., Santa Clara, CA, USA). Further enhancement was performed with the Tyramide Signal Amplification kit (Invitrogen, Waltham, MA, USA) and color development was performed with AEC (Zymed technologies, Waltham, MA, USA). The sections were counterstained with Mayer’s hematoxylin. Quantitative evaluation of the specific staining for FGF23 was performed with NIS Elements digital imaging software (Nikon, Tokyo, Japan). Digital images were made from the medial cortex of two sections of each tibia at 10x magnification. The area of positively-stained osteocytes, the area of positively-stained capillairy bloodvessels and the total bone area was measured. The percentage of positively-stained bone area and the percentage of positively stained vessel area were calculated. Data from the two sections of each tibia were averaged.