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

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-dihydroxytryptamine (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.

Number and Density of AMPA Receptors in Single Synapses in Immature Cerebellum

“…The usual photoconversion method (Lübke, 1993) uses radicals from fluorophores injected into cells by irradiation of excitation light. In contrast, this 2p photoconversion method uses radicals from not only fluorophores but also endogenous radical generators, probably NADPH and/or flavoprotein, which have two-photon absorption cross sections at 720 -800 nm (Xu et al, 1996;Huang et al, 2002) (see Results).…”

Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy.

“…The reaction product is highly insoluble and easily visible with ordinary transmitted light microscopy. Since the DAB reaction product is osmiophilic, this technique can be used for correlative light and electron microscopic studies using a variety of fluorescent compounds including Lucifer yellow, DiI, Bodipy ceramide, fluorogold, and fluororuby (Pagano et al, 1989;Bentivoglio and Su, 1990;yon Bartheld et al, 1990;Balercia et al, 1992;Lubke, 1993;Papadopoulos and Dori, 1993;Schmued and Snavely, 1993;Takizawa et al, 1993). A potentially powerful application of this technique is for correlated light and electron microscopic studies of immunolabeled specimens.…”

Multi-dimensional correlative imaging of subcellular events: combining the strengths of light and electron microscopy

“…Successful photoconversion of DAB has also been obtained by using ethidium bromide, tetramethyl rhodamine, Evans fast blue, acridine orange, and nuclear yellow (Lübke 1993). Rhodamine-dextran-amine (Fluoro-Ruby) has been exploited in labeling axons and has been used successfully to photo-oxidize DAB (Schmued and Snavely 1993).…”