JoVE Encyclopedia of Experiments
Biological Techniques
0 views • 5:00 min • July 8th, 2025
Begin Hematoxylin and Eosin staining by taking a glass slide containing the paraffin-embedded tissue section. Dip the slide in a deparaffinization solution containing xylene - an organic solvent that dissolves the paraffin layer around the tissue, making it accessible to stains in subsequent steps.
Treat the tissue section with absolute alcohol to remove the xylene. Immerse the tissue in decreasing concentrations of alcohol. This step rehydrates the tissue by replacing the alcohol with water for better compatibility with water-soluble dyes.
Stain the tissue with basic hematoxylin, which in its oxidized form binds to mordant - a metal cation. This complex electrostatically binds to the nucleic acids in the genome, staining the nucleus purple.
Submerge the stained section in an acidic differentiation solution to remove excess hematoxylin from the cytoplasm, making the dye-retaining nucleus appear distinct. Treat the tissue with an alkaline bluing agent to neutralize the free acids. This regresses the purple dye color to blue, enhancing the nuclear details.
Immerse the slide in acidic eosin solution to stain the basic protein components in the cytoplasm and the connective tissue fiber
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