Apr 21, 2023

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

DOI
https://dx.doi.org/10.17504/protocols.io.4r3l2718qg1y/v1
  • Donna Peehl1,
  • Renuka Sriram1,
  • Rosalie Nolley1
  • 1University of California, San Francisco
Rosalie Nolley
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DOI: https://dx.doi.org/10.17504/protocols.io.4r3l2718qg1y/v1
Protocol Citation: Donna Peehl, Renuka Sriram, Rosalie Nolley 2023. Bone Decalcification Protocol Using 14% EDTA Buffer Solution pH 7.2 - 7.4. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2718qg1y/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 09, 2023
Last Modified: April 21, 2023
Protocol Integer ID: 78391
Keywords: bone decalcification protocol, decalcification, successful decalcification, calcified tissue, minerals from bone, removing mineral, routine processing to paraffin, mineral, bone, quality paraffin section
Funders Acknowledgements:
NIH
Grant ID: U24CA253377
Abstract
This protocol describes the steps required for successful decalcification. Decalcification describes the technique for removing minerals from bone or other calcified tissue so that good-quality paraffin sections can be prepared that will preserve all the essential microscopic elements. Decalcification is carried out after the specimen has been thoroughly fixed and prior to routine processing to paraffin.








Materials

ABC
10% Buffered FormalinFisherSTL286001
Ethylenediaminetetraacetic (Acid free EDTA)VWR97061-404
Ammonium hydroxideFisher1336216
Phosphate Buffered SalineGibco10010-023









Preparation of Ethylenediaminetetraacetic acid (EDTA)
Make 14% EDTA solution fresh
a. Add 140 g free acid EDTA to 700 mL distilled H2O
b. On stir plate in the fume hood, add ammonium hydroxide, 30 mL at a time, until solution clears (about 90 mL total)
c. Add H2O to almost 1 L . Check pH and adjust with ammonium hydroxide dropwise up to pH 7.4, then adjust final volume to1 L

Procedure
3d
Dissect bone and remove as much soft tissue as possible.
After appropriate fixation in 10% buffered formalin - 24:00:00 -48:00:00 hours, wash tissue in distilled H2O or used EDTA solution.

3d
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.


Note
Fluid volume to tissue ratio is critical for the decalcification process.



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 00:30:00 minutes


30m
Rinse twice in ddH20 for 00:30:00 minutes

30m
Place in 70% Etanol (EtOH). Store at 4 °C (no more than 2 weeks)

Proceed with tissue processing