Rapid Extraction with Ethyl Acetate of Free Fluorescein Derivatives from Fluorescein isoCyanate-Globulin Conjugates

Abstract

THE technique of staining tissues specifically with fluorescein-Conjugated globulin has found wide application since it was introduced by Coons, Creech and Jones in 1942¹. Though Coons and Kaplan² have recently introduced technical improvements the procedure remains in part tedious and time-consuming. Conjugation between fluorescein isocyanate and protein is effected in a bicarbonate buffer (pH 9.0) in the presence of dioxan and acetone² ; conjugation, however, is never quantitative and the presence of free dye in the protein solutions is inevitable. The free fluorescein derivatives must be eliminated since, when present in tissue sections being viewed with the fluorescence microscope, they impart a hazy appearance. At present the free fluorescein derivatives are removed from conjugates mainly by exhaustive dialysis and afterwards by adsorption with acetone-extracted or lyophilized mouse liver preparations. The dialysis is time-consuming, while in our experience prolonged adsorption with organ preparations removes specific conjugates as is shown by a fall in the antibody titre.