A MULTILABELING TECHNIQUE FOR SIMULTANEOUS DEMONSTRATION AND QUANTITATION OF KI-67 AND NUCLEOLAR ORGANIZER REGIONS (AGNORS) IN PARAFFIN-EMBEDDED TISSUE

Although many investigators have demonstrated a relationship between a rgyrophilic nucleolar organizer regions (AgNORs) and Ki-67 expression in solid tumors, no previous studies have simultaneously assessed the relationship between AgNOR and Ki-67 expression in paraffin-embedded t issue. We describe a method for simultaneous demonstration and quantit ation of Ki-67 and AgNORs in routinely processed tissue. The Ki-67 equ ivalent monoclonal antibody MIB1, which can detect proliferative activ ity in routinely processed tissue with microwave heating, was employed . Fresh human tonsil tissue was fixed in formalin and embedded in para ffin for Ki-67/AgNOR dual staining. Image analysis was employed for qu antitation of AgNOR staining in Ki-67-positive and Ki-67-negative nucl ei. The double-staining procedure had no measurable effect on the indi vidual parameters: Ki-67 labeling index, mean AgNOR number (NN), and N OR percentage nuclear area (NPNA). However, microwave processing for K i-67 immunostaining significantly increased nuclear area (NA) and AgNO R area (AA). A significant difference was found between Ki-67-positive and Ki-67-negative cells for NN (p<0.001), NA (p<0.001), AA (p<0.001) , and NPNA (p<0.001). These results suggest a direct relationship betw een AgNOR and Ki-67 in paraffin-embedded tissue.