Differentiation Solution

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

• Desired staining patterns can be obtained by varying the degree of exposure to the differentiation solution
• Results are sharper and more rapid compared to other differentiation methods
• Crisp and clear differentiation between cellular structures is obtainable when performed according to Sigma-Aldrich Procedure No. GHS

In regressive hematoxylin staining, a highly concentrated hematoxylin solution is used to rapidly overstain both nuclei and cytoplasm, and the cytoplasm is subsequently decolourised 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.

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
Acidified alcohol solution for the differentiation of regressive hematoxylin stains