Deparaffinization and rehydration of the decalcified femurs was performed. Antigen unmasking was performed using a sodium citrate buffer (pH 6.0) and blocked in 1% bovine serum albumin (BSA) and 5% normal goat serum in PBS for 1 h. Primary antibodies, i.e., rabbit polyclonal osterix antibody (Santa Cruz, sc-22536-R), rabbit polyclonal osteopontin antibody (Abcam, ab8448), rabbit polyclonal osteocalcin antibody (Santa Cruz, sc-30044), or PDGFRβ antibody (Abcam, ab32570), in a 1% BSA solution were added; then, the mixture was incubated overnight at 4 °C (1:100). The secondary antibody used was an Alexa Fluor dye conjugated (1:500) in an appropriate blocking solution for 1 h at room temperature in the dark, and nuclei were counterstained with DAPI.
The fluorescence intensity was measured in arbitrary units (a.u.) from 10 sections and analyzed by confocal microscopy (Leica TCS SP8 confocal microscope).
VEGFR2 protein expression was evaluated by immunohistochemistry using a Novolink Polymer Detection Systems Novocastra (RE7280-K, Leica Biosystems) according to the manufacturer’s instructions. The sections were deparaffinized in Dewax solution (AR9222, Leica Biosystems) and rehydrated in alcohol solutions (100%, 95%, and 70%) and then exposed overnight to specific primary VEGFR2 antibody (dilution 1:200; ab2349). The Novolink polymer highlighted the protein VEGFR2 in the tissue.
The fluorescence intensity was measured in arbitrary units (a.u.) from 10 sections and analyzed by confocal microscopy (Leica TCS SP8 confocal microscope).
VEGFR2 protein expression was evaluated by immunohistochemistry using a Novolink Polymer Detection Systems Novocastra (RE7280-K, Leica Biosystems) according to the manufacturer’s instructions. The sections were deparaffinized in Dewax solution (AR9222, Leica Biosystems) and rehydrated in alcohol solutions (100%, 95%, and 70%) and then exposed overnight to specific primary VEGFR2 antibody (dilution 1:200; ab2349). The Novolink polymer highlighted the protein VEGFR2 in the tissue.