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.
Coons, A. H., Creech, H. J., and Jones, R. N., J. Immunol., 45, 159 (1942).
Coons, A. H., and Kaplan, M. H., J. Exp. Med., 91, 1 (1950).
Weiler, E., Z. Naturf., 11,b, 31 (1956).
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DINEEN, J., ADA, G. Rapid Extraction with Ethyl Acetate of Free Fluorescein Derivatives from Fluorescein isoCyanate-Globulin Conjugates. Nature 180, 1284 (1957). https://doi.org/10.1038/1801284a0
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