4.5. Immunohistochemical Analysis of Bone Biopsy Samples
Immunohistochemical analysis was performed on four histological bone biopsy samples to detect Osx transcription factor and BMP-2 protein. Bone tissue samples with a thickness of 5 μm were deparaffinized in xylene (BioGnost Ltd., Zagreb, Croatia) and then rehydrated in alcohol of decreasing concentration (100%, 95%, 85%, and finally 75%). The next step was protein renaturation, which was achieved by incubating the slices in citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) in a water bath at 90° for 10 min. After cooling, the samples were washed in a physiological solution buffered with phosphate-buffered saline (PBS) at pH 7.2. Blockade of endogenous peroxidase activity to avoid nonspecific binding was performed with 0.3% hydrogen peroxide H