Hematoxylin is a natural dye obtained from the wood of the Haematoxylum campechianum tree. The name is derived from Greek words haimatos, meaning blood, and from xylon, meaning wood. The commercial hematoxylin comes from this tree species, which can be found in Central and South America. Although it can be synthesized in the laboratory, the majority of commercial hematoxylin is still obtained from the trees. The wood is breaking down in small pieces and boiled. A reddish solution is first obtained, that later changes to yellow, and finally becomes dark as it gets cold. The water evaporates, leaving a fresh compound containing 10 % of hematoxylin. After that, it is necessary to purify the compound with ether, dry the solution, and finally let it to crystallize in water solution.
Hematoxylin was used for the first time by Böhmer (1865), who added a mordant, and Fischer (1875). Wossowzky (1876) proposed the double staining hematoxylin and eosin in the same protocol.
The dye is not the hematoxylin itself but the hematein, a molecule that results from the chemical oxidation of the hematoxylin (Figure 1). Hematein can also be obtained by letting the hematoxylin solution opened to the air during 6 to 8 weeks, during which it is oxidized by the air oxygen. The sodium iodate is the chemical oxidant most used (0.2 g of sodium iodate oxidizes 1 g of hematoxylin). Other chemical oxidants are iodine, hydrogen peroxide, mercuric oxide and potassium permanganate. The oxidation by atmospheric oxygen continues the chemical oxidation of the hematoxylin solution, and precipitates may appear by over-oxidation if the solution is too old, which yields bad staining. Over-oxidation can be reverted by adding glycerol or alcohol to the solution. Commonly, during the preparation of the hematoxylin solution, an amount of chemical oxidant is added so that it is able to oxidize half of the hematoxylin of the solution. In this way, the other half of hematoxylin is oxidized progressively over time by oxygen and the staining solution can be used for a long time. Hematein is not commercially sold in solution because it is unstable.
After adding sodium iodate, the staining solution can be boiled to accelerate the oxidation process. So much that the solution can be used as soon as it gets cold. The boiling is no necessary and the same result can be obtained by leaving the solution for several days at room temperature.
The solutions with hematein show a reddish color at pH lower than 1, yellowish at pH between 1 and 1.5, and purple at pH above 6. To be able to stain, hematein needs the help of negative charged ionic metals. These metals are known as mordants. The more common are aluminum salts, such as potassium and ammonium alum, or iron salts, such as iron chloride and iron alum. Solutions containing hematein and aluminum are called hemalum. Other less used mordants are chrome alum, or those containing lead, copper, zirconium, phosphotungstic acid or molybdic acid. The amount of metalic ions needs to be above the hematein in an acidified solution. The hematoxylin with aluminum mordant is used for visualization of nuclei, with iron mordants is used in strong acid solutions to visualize nuclei and muscle cell striations, with phosphotungstic to make more visible the muscle striations, fibrin and glial cell fibers. The final color of hematoxylin can be changed by using different mordants. Thus, the aluminum mordant provides a blue color, iron mordant leads to a nearly black color, and tin mordant turns the color to red. The solutions of hematoxylin and iron salts do not need the addition of extra compounds for hematoxylin oxidation, since the iron salts themselves work as both mordant and oxidizing agent.
Hematoxylin staining may be progressive or regressive. It is commonly preferred the progressive staining, that is, the intensity of color gets stronger with the staining time. If the section is over-stained, the intensity of the color can be reduced with an acid solution, but the process is slow. The speed of staining can be slowed down by adding more aluminum or lowering the pH. The hemalum changes the color from purple to blue at pH 6. That is the reason of a tap water step after the staining solution.
The hematein-iron solution provides a darker color. In the Heidenhain hematoxylin, the hematein and the iron are applied in successive steps, yielding a wide variety of stained structures by differentiation. This procedure is preferred when acid solutions are going to be used after the hematoxylin steps because the staining of the nuclei is very strong. In addition, it is a progressive staining.