Differentiation solution comprises acidified ethanol or isopropanol that is used to decolorize hematoxylin stain. The duration of exposure to the solution determines the degree of decolorization. Differentiation is a crucial step in hematoxylin-eosin staining to achieve the proper color contrast necessary for visualization, by selectively removing excess hematoxylin from the tissue section and leaving behind the desired staining intensity.

酸化的酒精溶液可用于区分退行性苏木精染色。

Differentiation solution is employed mainly for the differentiation of regressive hematoxylin stains. It has been used in the following studies:

• the prediction of pulmonary metastasis progression in osteosarcoma patients
• the development of multispectral imaging to detect melanin

In regressive hematoxylin staining, a highly concentrated hematoxylin solution is used to rapidly overstain both nuclei and cytoplasm, and the cytoplasm is subsequently decolorized and excess hematoxylin is removed from the nucleus with dilute acid. Improper differentiation leaves excess residual hematoxylin that obscures the fine details of nuclear structures and chromatin and prevents eosin uptake.