Metachromasia of 3-amino-9-ethylcarbazole (AEC) and its prevention in immunoperoxidase techniques.

3-Amino-9-ethylcarbazole (AEC) used as chromogen in immunoperoxidase techniques normally has an intense, red colour. However, as an inconstant phenomenon, a pale yellowish-green reaction product severely impairing the evaluation can be observed. In order to circumvent this undesired effect, factors such as tissue fixative, proteolytic digestion, antibody concentrations and incubation time of the primary antibody were analyzed. The most important factor inducing a change in colour is probably the inadequately high local peroxidase concentration arising as the consequence of high amounts of bound primary antibody. This high enzyme concentration might cause metachromasia of AEC by producing the yellowish-green quinone-di-imine form of the substrate. As could be shown by spectrophotometry in test tube experiments, AEC metachromasia was proven to be enzyme dependent. Thus, the best way to trigger the local enzyme concentration on a tissue section to adequate levels appears to be the dilution of the primary antibody.