Sections prepared for a special stain have been accidentally stained with hematoxylin. To remove the hematoxylin, place sections in

• A. Acid Alcohol
• B. Water
• C. Acetone
• D. Hydrogen Peroxide

Answer: (A)

Differentiation with acid alcohol is used to selectively remove hematoxylin from areas where it is not needed, while keeping the desired staining intact. Hematoxylin is a basic dye that binds to basophilic structures; when sections have been accidentally over-stained, acid alcohol lowers the pH and weakens the dye’s grip on non-target components, allowing the unwanted hematoxylin to be washed away. This leaves the portions intended for the special stain in the correct contrast for the subsequent steps.

Water would merely rinse without differentiating, acetone can act as a strong solvent that may disrupt tissue or remove stains unpredictably, and hydrogen peroxide is an oxidizing bleach that would alter or bleach dyes rather than serve as a controlled differentiating step.