If the blueing agent is not properly removed during H&E staining, the cytoplasm appears which color?

• A. Very pale pink
• B. Dark blue
• C. Orange
• D. Gray

Answer: (A)

In H&E staining, eosin provides the pink color for the cytoplasm, while hematoxylin colors nuclei blue-purple. The blueing step is done after hematoxylin to push the color toward a blue hue for the nuclei, and it should be washed away before applying the eosin counterstain. If the blueing agent isn’t properly removed, the tissue environment remains more alkaline when eosin is applied, which reduces eosin’s binding and staining intensity. The result is cytoplasm that appears very pale pink (instead of a strong pink).