Just like the regressive stain, it is preferred to start off with a basic protocol that is fairly easy to adapt to pathologists' preferences.
| Step | Time |
| Oven (if on stainer) | 15 mins |
| Xylene | 2 mins |
| Xylene | 2 mins |
| Xylene | 2 mins |
| 100% ethanol | 2 mins |
| 100% ethanol | 2 mins |
| 95% ethanol | 1 min |
| Water wash | 1 min |
| Hematoxylin | 3 mins |
| Water wash | 2 mins |
| Differentiator | 30 secs (for mild acids ONLY; works best on platform stainers) |
| Water wash | 1 min |
| Bluing | 1 min |
| Water wash | 1 min |
| 95% ethanol | 1 min |
| Eosin Y | 45 secs |
| 95% ethanol | 30 secs |
| 100% ethanol | 1 min |
| 100% ethanol | 1 min |
| Xylene | 2 mins |
| Xylene | 2 mins |
Remember, there is flexibility if you would like an additional xylene at the beginning and/or at the end prior to coverslipping. It has also been found that this protocol works well with just about any commercially-available hematoxylin or eosin. The differentiation step should be a solution made with a mild acid (eg, citric acid). The differentiator in this case is not intended to remove any stain from the nuclei, but is instead for removing any background staining or excessive staining of mucin.
