A 4-micron thick section is cut from the paraffin block. The section is stained with an immunoperoxidase method using the monoclonal antibody Ki-67 (MIB-1 clone). This is the paraffin nuclear epitope to the Ki-67 antigen. Any nucleus that has an antigen-antibody complex will cause the bright-field, brown chromogen, diaminobenzidine (DAB), to precipitate onto it. All nuclei, both DAB positive and negative, are counterstained with diluted hematoxylin.
Ki-67(MIB-1)-stained slides are scanned using the Aperio ScanScope instrument, which captures digital images of the patient slide. The technologist views the digitized image on a computer monitor and using a tracing tool available in ImageScope (Aperio Technologies Inc), traces around areas of invasive or metastatic cancer capturing no less than 75% of the total cancer within the image. The traced areas are then analyzed using an image analysis algorithm that renders the percentage of positive-staining tumor nuclei. The slides and test results are then reviewed by a pathologist who provides a final interpretation.(Unpublished Mayo method)