Quantifying Anti-Proliferative Effects

The anti-proliferative activity of test formulations was determined by tumor immunohistochemical staining of the Ki-67 proliferating protein in formalin-fixed, paraffin-embedded specimens (Yi et al., 2018 (link)). Briefly, 4 μm-thick tumor tissue sections were dried, deparaffinized, and rehydrated following standard procedures. Sections were subjected to heat-induced antigen retrieval. Immunohistochemical staining was performed using Ki-67 antibody (Monoclonal Mouse Anti-Human, Dako Agilent autostainer (Twinsburg, OH)). Optical densities of the Ki 67-positive areas in the tumor sections were measured and the % Ki-67 proliferative protein expression calculated. This was achieved by digital image analysis using Image J software (version 1.45s, Bethesda, MD) together with computer-assisted microscopy (El Sayed et al., 2018 (link)).