Tissue Decalcifying Solutions

Hard tissues such as bone, cartilage, and teeth, as well as calcified lesions and tissues containing stones, are too hard to slice into thin sections as they are. Decalcification is the process of removing calcium from hard or calcified tissue to allow sectioning. Fujifilm Wako offers a line-up of various tissue decalcifying agents. Kalkitox™, which is composed mainly of hydrochloric acid and EDTA, is a recommended decalcifying agent. It offers fast decalcification like acid decalcification, and can be used for immunostaining like chelate decalcification.

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Tissue Decalcifying Solutions

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Decalcification Methods Using Acids

In acid decalcification, the sample is soaked in a dilute solution of acid to decalcify. Nitric acid, trichloroacetic acid, and formic acid are commonly used, but mixtures such as Plank-Rychlo solution, consisting of aluminum chloride, hydrochloric acid, and formic acid, or Morse solution, consisting of formic acid and sodium citrate, are also used. The properties of each are summarized below.

Acid	Property	Notes
Nitric acid (5-7.5%)	Fast decalcification	Neutralization with 5% sodium sulfate is necessary to prevent tissue swelling. Prolonged decalcification results in significant loss of stainability.
Trichloroacetic acid (5%)	Fast decalcification Preserves stainability of the nucleus	Neutralization with 5% sodium sulfate is necessary to prevent tissue swelling.
Formic acid (1-5%)	Fast decalcification Little or no loss of stainability Little or no tissue shrinkage	－
Plank-Rychlo solution	Extremely fast decalcification (3 times faster than nitric acid or formic acid)	Insufficient neutralization and washing will greatly reduce stainability. Neutralization with 5% sodium sulfate is necessary to prevent tissue swelling.
Morse solution	Tissue swelling is unlikely to occur. Can be used for immunostaining and in situ hybridization (comparable to EDTA decalcification)	Slow decalcification (3 times slower than 5% nitric acid, though 8 times faster than decalcification by EDTA) Neutralization with 5% sodium sulfate is necessary to prevent tissue swelling.

Decalcification Methods Using Acids Chelating Agents

Decalcification with chelating agents such as EDTA is used in immunostaining and in situ hybridization (ISH), because it preserves immunogenicity and enzyme activity. It also has little effect on tissue structure, making it suitable for microstructural observation. On the other hand, slow decalcification is a disadvantage, as several weeks are required to decalcify.

References

“Handbook for Staining and Bioimaging Experiments 5th ed.” ed. by Takata, K., Saito, N. and Kawakami, H., Yodosha, Japan, (2012). (Japanese)
Okubo, K.: “Master of cell and tissue staining”, Yodosha, Japan, (2018). (Japanese)

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