The hematoxylin (in Greek haimatus: blood and xylon: wood) is a natural compound from the leguminous Haematoxylum campechianum. The hematoxylin has to be oxidized into hematein to be used as a dye, and then combined with a metal, which works as a mordant. The hematoxylin oxidation may be oxidant substances or by the air oxygen, that takes longer. It is the hematein the dye that shows affinity for acid cellular components, such as the chromatin of the nucleus. The hematoxylin-eosin staining is probably the most widely used in histology labs as a general staining technique. Hematoxylin is also part of other staining protocols, as in the thrichrome procedures for staining nuclei.
Weigert's hematoxylin, or ferric hematoxylin, stains nuclei, and it is choose when an acid solution is used in a later step of the protocol. The hematoxylin is combined with a mordant, such as potassium aluminum or ammonium aluminum. That is why it resists acid solutions. In the Weigert's heamtoxylin staining, the mordant and the dye are stored separately, and joined just before the staining. The final color of the nuclei ranges from black to dark purple. Weigert's hematoxylin is part of the van Gieson's trichrome staining procedure.
Procedure
Solution A (Hematoxylin)
1 g hematoxylin (C.I. 75290)
100 ml 96º ethanol
Solution B (Iron chloride)
1.16 g iron chloride (III)
1 ml of 25 % of hydrochloric acid
99 ml distilled water
Notes
The two components (solution A and B) are stored separately and mixed before use. The proportion is 1:1 (for example, 100 ml of solution A and 100 ml of solution B). The staining time depends on the technique and the tissue.
After the hematoxylin staining, and after thorough rinses in tap water (about 30 min), sections can be immersed in 0.1 % of sodium bicarbonate to get a bluish color of the nuclei. The time in this solution depends on the final color we want to get. It can be controlled under the microscope.
Products
Hematoxylin (C.I. 75290)
Iron chloride (III)
Hydrochloric acid
96º ethanol
Distilled water
Labware
Test tubes
Balance
Magnetic stirrer
Flasks
Bottles