VECTOR ALKALINE-PHOSPHATASE SUBSTRATE BLUE-III - ONE SUBSTRATE FOR BRIGHTFIELD HISTOCHEMISTRY AND HIGH-RESOLUTION FLUORESCENCE IMAGING BY CONFOCAL LASER-SCANNING MICROSCOPY

A number of histochemical chromogenic substrates for alkaline phosphat ase are commercially available and give reaction products with a range of colours for brightfield examination. Some of these reaction produc ts are also fluorescent, exhibiting a wide excitation range and a broa d emission peak. We report here that one of these substrates, Vector B lue III, yields a stable, strongly fluorescent reaction product with a n excitation peak around 500 nm and a large Stokes shift to an emissio n peak at 680 nm. The reaction product can be excited using a mercury lamp with a fluorescein excitation filter or an argon ion laser at 488 nm or 568 nm, and the emission detected using a long-pass filter desi gned for Cy-5. Thus, a single substrate is suitable for brightfield im aging of tissue sections and high-resolution analysis of subcellular d etail, using a confocal laser scanning microscope, in the same specime n. (C) 1998 Chapman & Hall.