Bone Decalcification Protocol Using 14% EDTA Buffer Solution pH 7.2 - 7.4

Make 14% EDTA solution fresh

a. Add 140g free acid EDTA to 700mL distilled H2O

b. On stir plate in the fume hood, add ammonium hydroxide, 30mL at a time, until solution clears (about 90mL total)

c. Add H2O to almost 1L. Check pH and adjust with ammonium hydroxide dropwise up to pH 7.4, then adjust final volume to1L

Dissect bone and remove as much soft tissue as possible.

After appropriate fixation in 10% buffered formalin - 24h 0m 0s-48h 0m 0s hours, wash tissue in distilled H2O or used EDTA solution.

Place tissue in 14% EDTA solution at 4°C C on a stirring device to circulate the EDTA. Use enough solution to saturate tissue (fluid volume to tissue ratio is critical for the decalcification process). The EDTA solution should be at least 20X the volume of the tissue to ensure proper decalcification.

Periodically check bone for adequate decalcification. Refresh EDTA solution daily for first 5 days, then may leave in same EDTA solution without changing.

Decalcification is complete when bone is soft and pliable. This may take 10 days or more depending on tissue size. Can check by X-ray, or probe with a needle and/or bend tissue to determine if tissue is soft enough to section. Over-decalcification will cause tissue or cells to lose affinity for certain stains.

Rinse once with Phosphate Buffered Saline (PBS) for 0h 30m 0s minutes

Rinse twice in ddH₂0 for 0h 30m 0s minutes

Place in 70% Etanol (EtOH). Store at 4°C (no more than 2 weeks)

Proceed with tissue processing