Flazo Orange for Masking of Auto and Nonspecific Fluorescence of Leukocytes and Tissue Cells

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Abstract
Flazo orange (Hall and Hansen 1962, Zbl. Bakt., 1 Orig., 184: 548-54) was tested on several types of tissue and leukocyte preparations. Cells were simultaneously mixed with bacteria for which homologous sera labeled with fluorescein isothiocyanate had been prepared. The slides were treated with the fluorescent antibody followed by Flazo orange as a counterstain which was found constantly to overcome the auto and nonspecific fluorescence of tissue and lymphocytic cells while allowing the specific green fluorescence of the bacteria to be observed. The granulocytic cells still exhibited a high nonspecific fluorescence which could be eliminated by a 30 sec treatment of the preparation with 0.1 N HCl. The specific fluorescence remained observable although diminished in intensity. Tissue culture coverslips of Chang's conjunctiva cell line were treated with labeled normal rabbit serum and counter-stained with Flazo orange. Both auto and nonspecific fluorescence were masked.