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The removal of calcium deposits is essential for good embedding procedure. Decalcification is usually carried out between the fixation and processing steps (Section 1.3). Bone must obviously be processed in this way, but other tissues may also contain calcified areas. A variety of agents or techniques have been developed to decalcify tissues, each with advantages and disadvantages. Immersion in solutions containing mineral acids, organic acids, or EDTA are the predominant methods used. Electrolysis has also been tried.
Strong mineral acids such as nitric and hydrochloric acids are used with dense cortical bone because they will remove large quantities of calcium at a rapid rate. As might be expected, these strong acids also damage cellular morphology. Mineral acid decalcifiers are not recommended for delicate tissues such as bone marrow. Because they are not as aggressive, organic acids such as acetic and formic acid are better suited to bone marrow and other soft tissues. Organic acids act more slowly than mineral acids, and will require extended treatments to decalcify cortical bone. Formic acid in a 10% concentration is the best all-around decalcifier. Some commercial solutions combine formic acid with formalin to fix and decalcify tissues at the same time.
EDTA can remove calcium and is not as harsh as mineral or organic acids. The use of EDTA is limited by the fact that it penetrates tissue poorly and works slowly. It is also expensive in large amounts. Electrolysis has been tried in experimental situations where calcium had to be removed with the least tissue damage. Electrolysis is slow and not suited for routine daily use.
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