As mentioned earlier, differentiation is used with regressive staining techniques and is used to remove the excess dye that has been taken up 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 a problem.