As mentioned earlier, differentiation is used with regressive staining techniques to remove the excess dye that has been absorbed into the tissue until a crisp delineation of the desired parts is achieved.
Basic dyes are differentiated using a weakly acidic solution prepared with alcohol.
Acid dyes are differentiated using a weakly basic solution prepared with alcohol.
Aqueous-based differentiating solutions are not recommended as the dyes will diffuse more rapidly, and over-differentiating becomes problematic.
