Development of a robust and reproducible MQIF assay to assess Menacalc. A. Representative tumor histospot with Pancytokeratin staining used to define the tumor mask, pan-Mena (red) and Mena11a (green) showing a cytoplasmic staining pattern. Scale bar is shown at the left bottom of the panels. B, C. Reproducibility of MQIF Menacalc assay. Linear regression between two independent runs of pan-Mena (B) and Mena11a (C) multiplexed on serial sections of an index TMA have a high correlation (R2 = 0.84 and 0.73 respectively). D. Linear regression between pan-Mena and Mena11a on an index TMA slide shows no correlation (R2 = 0.06).