Decalcification Protocol

• Decalcification is required for processing bone tissue in routine diagnostic practice. Control of this step is crucial because it may have important consequences for establishing the diagnosis.
• Agents commonly used for decalcification are acids of varying ionic strength or chelating agents. Acids that ionize and solubilize calcium ions include strong inorganic acids, such as hydrochloric or nitric acid, and weaker organic acids such as formic or phosphoric acid. Chelating agents such as ethylenediaminetetraacetic acid (EDTA) link to calcium ions and form an insoluble complex.

Routine Decalcification

• Make 14% EDTA solution. Add 140 g free acid EDTA to 700 ml distilled H₂O. On stir plate in the fume hood, add ammonium hydroxide 30 ml at a time until solution clears (about 90 ml total). Add H₂O to almost 1 L. Check pH and adjust with ammonium hydroxide dropwise up to pH 7.2, then adjust final volume to 1 L.
• Place fixed / rinsed tissue in at least 15 volumes of 14% EDTA, and change daily or 5x / week (M-F) with mixing, this means at least 15 ml for a mouse tibia / femur. Placing tissue in a screw-top tube on its side, on a rocker works well. We tape tubes into a rack and place the whole thing on its side on the rocker.

• Time of decalcification varies with tissue size, species, etc. Mouse adult long bones and vertebrae need 10-14 days, and isolated calvaria needs 2-4 days.
• Rinse in H₂O x 4. For paraffin embedding, place in 30%, 50%, and 70% ETOH for at least 30 min each prior to submitting to core lab; For frozen sections, blot tissue dry and freeze in OCT.

Hurry-Up Decalcification

• Reagent preparation. We need prepare 12.6 g aluminum chloride (hydrous), 8.5 ml 10 N hydrochloric acid, 5.4 ml 88% formic acid.
• Make final volume to 100 ml. Dilute 1 part of this stock solution with 4 parts of dH₂O for LM histology (or immunohistochemistry) and 1:8 for tissues being processed for EM.
• Decalcification of an entire head of a 100 g rat takes only about 5 days in this solution at 1:4 dilution. It is truly amazing how quickly it works and how reasonable the tissues look afterwards.
• We usually do the decalcification at 4°C as with EDTA (with stirring, large fluid to tissue ratio). You should change this solution daily. Wash the tissues liberally after decalcification as with EDTA.

• The fluid volume to tissue ratio is very critical for the decalcification process. It should be as large as practical, for example, no more than 10 or 15 hemi-jaws per liter of solution.
• You should change the solution every other day, pre chill the new batch to keep temperature at 4°C at all times.
• It is important to clean away as much muscle and soft tissues from the hemi-jaw bones, excess tissues act as a barrier to the decalcification process.

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