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Immunostaining to determine osteocyte apoptosis by caspase‐3

Caspases are cysteine proteases that play a critical role in apoptosis. Activated caspase‐3 is a downstream cell death signal. Thus, immunostaining by caspase‐3 can be used as an effective tool for the assessment of osteocyte apoptosis. The specific intent of this work was to optimize caspase‐3 immunostaining protocol. Working antibody dilution, method of epitope retrieval, peroxidase inactivation, and blocks were examined through various series of antibody runs. 1:300 appears to be a good working dilution for the caspase‐3 antibody. It produced a high signal to noise ratio with minimal nonspecific expression. Of the retrieval techniques analyzed, DeCal retrieval appeared to limit nonspecific staining the most. We have yet to determine the significance of peroxidase inactivation with 3% H2O2 in methanol. Casein appears to be a better blocking agent than serum. Caspase‐3 immunostaining is a vital tool in the evaluation of apoptosis. By considering the previously mentioned variables, we hope to have a protocol for a more accurate measure of osteocyte apoptosis.