The difference between methods is largely one of convenience. Progressive hematoxylins are generally less concentrated and work slowly to avoid overshooting the endpoint. Also, timing is not so important in regressive procedures. As long as the slide is overstained, it doesn't matter whether it was in the staining dish for 1 minute or 10. Regressive procedures are therefore faster and more convenient than the progressive ones, and they have the added advantage that differentiation also removes hematoxylin from the gelatin or other slide adhesive, producing a clear, transparent background.
Differentiation is stopped immediately by simply washing the slides in water.
If too much hematoxylin has been removed, a few more seconds in the stain will correct the problem, and the whole process can be repeated. If too little has been removed, a few more dips in acid alcohol will produce the correct endpoint. In a properly stained slide, only the nuclei, flecks of calcium, and certain kinds of mucus will be stained, while the background will be unstained or very pale brown. There should be no hint of blue or gray. The blueing step Because most alum hematoxylin formulae are fairly acid, the nuclei will at first be stained the purplish color of the acid dye.