Alkaline phosphatase cytochemistry in confocal scanning light microscopy for imaging the bone marrow stroma

This paper reports on the use of alkaline phosphatase cytochemistry and combined conventional and confocal reflection and fluorescence scanning light microscopic modes in the study of human marrow stroma. It was found that the end product of the enzyme reaction using Napthol AS phosphate as substrate and Fast Blue BB as coupler reflected the 633 nm (red) light from a Helium-Neon laser. Serial optical sections suitable for 3-D reconstruction and selectively depicting the marrow reticulum cells could be obtained from thick glycol methacrylate sections reacted for Alkaline phosphatase. Furthermore, the yellow background of uncoupled diazonium salt over cytochemically unreactive structures in the same specimens and fields was used for imaging haemopoietic cell mass by operating the microscope at 488 nm (argon ion laser, blue-green). These methods may offer advantages in the investigation of the bone marrow stroma and its interplay with haemopoiesis and osteogenesis in normal and disease conditions.