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 argyrophilic 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 tissue. We describe a method for simultaneous demonstration and quantitation of Ki-67 and AgNORs in routinely processed tissue. The Ki-67 equivalent monoclonal antibody MIB1, which can detect proliferative activity in routinely processed tissue with microwave heating, was employed. Fresh human tonsil tissue was fixed in formalin and embedded in paraffin for Ki-67/AgNOR dual staining. Image analysis was employed for quantitation of AgNOR staining in Ki-67-positive and Ki-67-negative nuclei. The double-staining procedure had no measurable effect on the individual parameters: Ki-67 labeling index, mean AgNOR number (NN), and NOR percentage nuclear area (NPNA). However, microwave processing for Ki-67 immunostaining significantly increased nuclear area (NA) and AgNOR 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 between AgNOR and Ki-67 in paraffin-embedded tissue.