A new immunofluorostaining method using red fluorescence of PerCP on formalin-fixed paraffin-embedded tissues

Abstract

Immunofluorostaining, a versatile tissue staining method, is used in biomedical research because of its clear contrast and precise quantification of positive signals. However, its application in clinical diagnosis has been limited. A major obstacle is high fluorescent background of formalin-fixed, paraffin-embedded tissue sections (paraffin sections). On paraffin sections, strong and broad fluorescence of the section overlapping that of conventional fluorescent dyes such as fluorescein isothiocyanate (FITC) prevents detection of target immunofluorescence. To circumvent the background, we selected an albuminous dye, peridinin chlorophyll a protein (PerCP), for immunostaining of human tumor sections with tumor-reactive monoclonal antibodies. Red fluorescence of PerCP clearly distinguished the tumor region within the yellow-green autofluorescence of the section. Furthermore, it was possible to observe tissue morphology simultaneously without any counterstaining; autofluorescence served as counterstaining in this method. Digital quantification of PerCP-stained image intensity correlated (r2>0.99) well with extracted PerCP amount, indicating the usefulness of image quantification. We conclude that this new and simple immunofluorostaining method can be applied to pathological diagnosis of a wide range of conditions, including cancer.