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The page below is a sample from the LabCE course Troubleshooting Guidance for Hematoxylin and Eosin (H&E) Stain. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Well-Rounded, Standard H&E - Regressive Protocol

When working with a new laboratory, I prefer to start them off with a basic protocol that is fairly easy to adapt to pathologists' preferences.
Oven (if on stainer)15 mins
Xylene2 mins
Xylene2 mins
Xylene 2 mins
100% ethanol2 mins
100% ethanol2 mins
95% ethanol1 min
Water wash 1 min
Hematoxylin3 mins
Water wash2 mins
Differentiator1 min (for mild acids ONLY; works best on platform stainers)
Water wash 1 min
Bluing1 min
Water wash1 min
95% ethanol1 min
Eosin Y45 secs
95% ethanol30 secs
100% ethanol1 min
100% ethanol1 min
Xylene2 mins
Xylene2 mins
Remember, there is flexibility if you would like an additional xylene at the beginning and/or at the end prior to coverslipping. I have also 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), instead of the traditional hydrochloric acid (HCl). While you can use strong acids on your instrument, remember that they can corrode the stainless steel components, in addition to being difficult to program. One second when staining by hand is much faster than what is experienced on a platform simply because the speed of arm movement must be considered. Just the movement time into and out of the reagent can add a few additional seconds, which may decolorize your tissue more than you intended.