The importance of optimal immunostaining conditions on the accuracy of ImmunoRatio analysis. The red lines outline the nuclei and highlight the segmentation of (a to c) brown and (d to f) blue staining components. (a) Overly dilute primary antibody concentration (Ki-67 MIB-1, 1:400) causes inadequate brown segmentation. (b) Optimal antibody dilution (1:100). (c) Overly strong antibody concentration (1:25) results in excessive cytoplasmic staining and brown segmentation. (d) Overly dilute hematoxylin staining causes inadequate blue segmentation. (e) Optimal hematoxylin dilution. (f) Overly strong hematoxylin causes excessive cytoplasmic staining and blue segmentation.