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 bones and teeth, and does not normally appear in a solid state in the tissues, but in some cases it precipitates as a solid and settles in the tissues, and this is called pathologic calcium deposition. 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 can form chelates with calcium, including alizarin red S, red violet, and nuclear fixation red; these dyes are generally better at recognizing moderate to large amounts of calcium than lightly stained trace calcium deposits, but alizarin red S tends to give more reliable results with small amounts of deposits.

Commonly used methods for calcium salt staining include silver nitrate and alizarin red S. Calcium salt staining solution (polychromatic methylene blue method) uses a combination of alizarin red S and polychromatic methylene blue for staining, with bone and alizarin red combining to form a red precipitate, cartilage reacting with polychromatic methylene blue to appear purple, with the cytoplasm appearing yellow, and the nucleus blue, making this the best staining method for the phalanx of a 6- to 8-month-old fetus.