The differentiation of stains allows for the ability to selectively remove stain from tissues to the preference of the viewer. Differentiation is a procedural staining step related to a regressive H&E staining method. When staining with hematoxylin, hydrochloric acid (for rapid differentiation) and acetic acid (for slower, more controlled differentiation) are the most commonly used components of differentiating solutions. While hydrochloric acid (HCl) has historically been the standard for differentiation, milder acids, such as acetic acid are being used more commonly to provide gentler dye removal. Part of this trend is due to the use of automated staining instrumentation, which must accommodate potential delays due to mechanics in addition to the time slides spend in the differentiating solution.
