Fluorescent protein tracers; a trial of new fluorochromes and the development of an alternative to fluorescein

Abstract

With the object of simplifying the fluorescent protein tracer technique, the following fluorochromes were examined as possible alternatives to fluorescein: aminoeosin, aminorhodamine B, 3-phenyl-7-isocyanatocumarin (Geigy), 5-β-carboxyethylaminoacridine, R 4388 (Geigy), fluolite C (I.C.I.), lissamine flavine FFS (I.C.I.), lissamine rhodamine GS (I.C.I.), and lissamine rhodamine B 200 (I.C.I.) (RB 200). With the exception of RB 200, none was suitable as a protein label largely because of unsatisfactory fluorescence intensity or colour.

RB 200 has proved a successful alternative to fluorescein. The conjugation of dye to protein by a sulphonamido linkage is quick and simple and does not materially affect the physico-chemical or biological properties of the protein. The resulting conjugates are stable, have a brilliant orange fluorescence in ultraviolet light and good contrast with tissue autofluorescence. The contrast is sufficient to permit the use in microscopy of ultraviolet plus blue light with a yellow filter above the object to ensure a black background; fluorescence is greatly enhanced in this way.

When injected intravenously into rats or rabbits, conjugates are distributed in the tissues and eliminated from the plasma in much the same way as proteins labelled with fluorescein or radio-active isotopes. Serum antibody conjugated with RB 200 retains immunological specificity as demonstrated by the staining of the corresponding antigen. Practical use has been made of RB 200 conjugates as plasma tracers and as specific immunological stains: they have been applied alone and in combination with fluorescein conjugates in double tracing experiments.