Isolation and Characterization of Fluoro‐Jade B, a Selective Histochemical Stain for Neuronal Degeneration

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Abstract
Fluoro‐Jade B is a novel fluorescent dye, and since its introduction in 1999 it has been widely used in neuroscience research for selectively staining degenerating neurons in brain tissue sections. However, the chemical composition of Fluoro‐Jade B has not been previously resolved. We here report successful separation and identification of eight isomers and structural analogues of Fluoro‐Jade B. Two analytical HPLC methods, consisting of a reversed‐phase C18 column and a mobile phase with either a pH gradient or an acetonitrile gradient, were developed. A quantitative separation was performed by a semi‐preparative reversed phase C18 HPLC column. Each individual component was characterized by LC/ESI mass spectrometry and NMR spectroscopy. The compounds 5‐(6′‐hydroxy‐3′‐oxo‐3H‐xanthen‐9′‐yl)benzene‐1,2,4‐tricarboxylic acid, 2‐(6‐hydroxy‐3‐oxo‐3H‐xanthen‐9‐yl)‐5‐(2,4‐dihydroxybenzoyl)terephthalic acid, and 4‐(6‐hydroxy‐3‐oxo‐3H‐xanthen‐9‐yl)‐6‐(2,4‐dihydroxybenzoyl)isophthalic acid represent three new fluorescent compounds discovered in Fluoro‐Jade B. They are, presumably, responsible for the dye's ability to detect degenerating neurons.