Calcium is present in large quantities in the human body, constituting the skeleton as a scaffolding to support the body, and also plays an important role in secretion, transportation, muscle contraction, and nerve conduction. Calcium exists in two forms in the body, one is ionized calcium, which exists within the blood circulation and is known as blood calcium, and the other is bound calcium, which is bound to proteins, carbonic acid, or phosphoric acid and deposited in the tissues. Calcium normally permeates all tissues and cells except the bones and teeth, and does not normally appear in the tissues in a solid state; however, in some cases where calcium precipitates as a solid and settles in the tissues, it is pathologically deposited as calcium salts. The deposited calcium salts are primarily calcium phosphate and to a lesser extent calcium carbonate, which is usually monorefractive, but calcium oxalate is birefractive. When stained with HE calcium generally appears as a purplish-blue color, and many dyes form chelates with calcium, including alizarin red S, red violet, and nuclear fixation red, etc. In general these dyes are better at identifying moderate to large amounts of calcium than are trace calcium deposits with light staining, but alizarin red S tends to give more reliable results with small amounts of deposits.

Commonly used methods for calcium salt staining include the silver nitrate method and the alizarin red S method. Calcium salt staining solutions use a combination of alizarin red S and solid green for staining. Alizarin red S is an anthraquinone derivative, a sodium salt of alizarin sulfonate, which chelates with calcium salts in calcium carbonate or calcium phosphate to form an orange-red complex; the calcium salts combine with the alizarin red to form a red-orange precipitate, which is suitable for staining small quantities of calcium-salt tissues.