Cell cultures were fixed for 30 minutes at room temperature with 4% paraformaldehyde. After washing with PBS, cells were permeabilized for 10 minutes with 10% (v/v) Triton X-100 in PBS, then washed twice with a serum wash containing 1% (v/v) sodium azide, followed by a blocking step containing 0.5% (v/v) Triton X-100 and 1% (v/v) sodium azide for 30 minutes at room temperature. Dilution buffer containing 2 ^g/ml polyclonal rabbit anti-human osteocalcin (Santa Cruz Biotechnology, Santa Cruz, CA) was added and incubated at 4°C overnight. Following incubation, the cells were rinsed twice with serum wash and incubated in the dark with 8 |g/ml FITC-labeled secondary antibodies for 30 minutes and counterstained with DAPI. The cells were then washed with PBS and fluorescence was observed using a Nikon Eclipse TE3000.
Results were evaluated using the student t test. Statistical significance was set at the 95% confidence level with a p-value < 0.05.
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