Photoconversion of diaminobenzidine with different fluorescent neuronal markers into a light and electron microscopic dense reaction product

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Abstract
This article describes methods for photoconverting diaminobenzidine (DAB) into a stable, light and electron microscopically visible dark reaction product in neurons which contain a fluorescent dye. Photoconversion of DAB has been achieved so far with the following fluorescent dyes: rhodamine labeled latex microspheres (RLM), 4,6‐diamidino‐2‐phenylindole (DAPI), 5,7‐di‐hydroxytryptamine (5,7‐DHT), Fast Blue (FB), Nuclear Yellow (NY), Diamidino Yellow (DY), Evans Blue (EB), acridine orange (AO), ethidium bromide (EBR),1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindolcarbocyanine perchlorate, D‐282 (DiI), propidium iodide (PI), and intracellularly injected Lucifer Yellow (LY). The dye is introduced into the neurons by tinctorial staining, retrograde transport, or intracellular injection. Photoconversion is conducted by incubating the tissue with the fluorescent substance‐containing cells in a DAB solution under simultaneous strong illumination with ultraviolet (UV) light. During the formation of the reaction product, the fluorescence disappears from the cell. In all cases, photoconversion provided a stable, nonfading DAB reaction product for light microscopy. In addition, at the electron microscopic level, it appeared that the photoconversion results in a homogeneously distributed, fine granular, dark, intracellularly located reaction product. With most of the retrograde tracers tested, photoconversion led only to staining of the cell bodies and the proximal portions of primary dendrites. Following photoconversion with intracellularly LY‐filled neurons and cells labeled retrogradely with DiI, DiO, and 5,7‐DHT, the reaction product was present throughout the cells, extending from the cell bodies into dendrites and dendritic appendices, and into axons. The high selectivity and methodological simplicity of photoconversion of DAB with fluorescent dyes into a stable, light and electron microscopical dense reaction product provide a promising alternative to classical neuroanatomical techniques and a new useful application of fluorescent neuronal tracers to light and electron microscopy.