The hardness of bone is due to calcium compounds. It is also frequently found in soft tissues. Small deposits, for instance, are viewed as an indicator of a possible malignancy in breast biopsies. It is often encountered in coronary arteries. Calcium deposits are very hard and are destructuve to knife edges, so they must be removed prior to sectioning. This process is called decalcification and is done mostly with diluted acids (formic, nitric), although chelating agents such as EDTA (ethylene diamine tetracetic acid) may be used when the effects that acids have on tissues must be avoided.
The commonest way of demonstrating calcium deposits is with silver nitrate using the von Kossa technique. The sections are placed into a silver nitrate solution and exposed to sunlight. This causes the silver to be deposited onto the anion associated with the calcium. Although calcium itself is not directly demonstrated with this technique, it is neverthless considered highly selective for calcium, since the anions which are blackened are invariably the calcium salt. Calcium itself can be demonstrated with the McGhee-Russel method using alizarin. Related dyes such as purpurin may also be used. Calcium deposits can also mordant hematein, and it often appears coloured purple-blue in H&E stained sections.