Simultaneous demonstration of CD15 and alkaline phosphatase activity in cryostat sections of rat fetuses: a detailed technical description for the developing brain.

CD15 and alkaline phosphatase are significant markers of the developing central nervous system. CD15 is known for its interaction in cell-cell contacts, while the presence of alkaline phosphatase is related to the formation of neuronal connections. This paper describes a combined immunocytochemical and enzyme histochemical technique to demonstrate both embryonic markers in the same cryostat section. The monoclonal antibody 3B9, which was used for the detection of CD15 recognizes the immunodominant carbohydrate structure alpha-1,3-Fucosyl-N-Acetyl-Lactosamine also known as X, LewisX, FAL or SSEA-1. This antigen dissolves and is easily rinsed out of the section. The procedure starts off with the detection of CD15. The results of the immunocytochemical procedure is a reaction product which is stable at a high pH, preventing loss of the immunocytochemical reaction product during the subsequent alkaline phosphatase detection. The other embryonic marker, alkaline phosphatase, can only be demonstrated enzymehistochemically if the enzyme is still active. The immunocytochemical localization procedure decided for, does not inactivate alkaline phosphatase totally. From the colorimetrical and histochemical alkaline phosphatase determinations it was concluded that the residual alkaline phosphatase activity detected with this technique, could be intensified by adding Mg(2+)-ions to both the colorimetrical and histochemical incubation media.