Abstract: Provided are a pretreatment method for detecting the number of cells containing Ki-67 protein-positive nuclei using Ki-67 antibody; a method for the detection; a kit to be used in the detection method; and determination of a therapy regimen using the aforesaid method. Attempts were made to activate Ki-67 antigen with the use of an enzyme having been considered as inappropriate for the activation thereof. By pretreating a sample with an enzyme not recognizing the epitope of MIB-1 (a rare cutter enzyme), the antigen activation of Ki-67 antigen was enhanced, while enhancing the antigenicity of other antigens including a cytokeratin too. As a result, a method for more objectively and more universally quantifying Ki-67-positive cells at higher reproducibility, said method comprising isolating cell nuclei from an FFPE section while enhancing the antigenicity and performing a reaction between Ki-67 protein, i.e., the target, existing in the cell nuclei with a fluorescently labeled antibody, has been completed.