Reduced Nonspecific Fluorescence of Paraffin Sections by Use of a Novel Red-Emitting Dye

Abstract

Green-emitting dyes such as fluorescein are relatively unsatisfactory for immunofluorescent labeling of sections of formalin fixed, paraffin embedded tissues because of nonspecific fluorescence associated with blue excitation. We have employed a novel red-emitting fluorochrome, designated Alexa 568, for immunohistochemistry on paraffin sections. Excellent results were achieved with this brightly fluorescent dye, including labeling of leukocytes in inflammatory lesions and of immunoglobulin or complement deposits in renal glomeruli. Minimal autofluorescence or formalin- induced fluorescence was observed using green excitation. Moreover, Alexa 568 exhibited greater photostability than fluorescein. Because fluorescent labeling is able to reveal greater detail, immunostaining of paraffin sections using this technique may be a useful alternative to enzymatic methods that generate an opaque, insoluble reaction product. In addition, this fluorochrome is likely to be especially useful for confocal laser scanning microscopy on paraffin sections, facilitating high-resolution imaging, quantitative studies, and three-dimensional reconstruction. (The J Histotechnol 23:99, 2000)