In hematoxylin dye solution, the chromatin in the nucleus is mainly deoxyribonucleic acid (DNA). In the double helix structure of DNA, the phosphate groups on the two chains are outward, negatively charged and acidic, which is easy to be dyed by ion or hydrogen bond interaction with the positively charged hematoxylin basic dye. Hematoxylin is blue in alkaline solution, so the nucleus is dyed blue.

Differentiation: After hematoxylin staining, the dye solution that does not bind to the cell is washed away with water, but the dye that binds to the cell nucleus too much and the dye adsorbed in the cytoplasm must be removed with the differentiation solution 1% hydrochloric acid alcohol to ensure the clear staining of the cell nucleus and cytoplasm. This process is called dyeing differentiation.

Because acid can destroy the quinone structure of hematoxylin, the pigment can be dissociated from the tissue, and the differentiation should not be excessive.

Blue: After differentiation, hematoxylin is in red ion state under acidic conditions and blue ion state under alkaline conditions, so after differentiation, it is washed with water to remove acid and stop differentiation, and then the nuclei stained with hematoxylin turn blue with weak alkaline water, which is called blue. Generally, it can turn blue when soaked in tap water, or it can turn blue with warm water (50 degrees warm water is the best).