A Good Baseline Staining Protocol

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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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A Good Baseline Staining Protocol

A mid-range stain is often preferred. The protocol below is utilized by the author when setting up a new laboratory or when helping to optimize an existing stain that has become less then optimal.
Reagent / Step Time
Xylene 2 minutes
Xylene 2 minutes
Xylene2 minutes
100% ethanol2 minutes
100% ethanol 2 minutes
95% ethanol2 minutes
Water wash2 minutes
Hematoxylin3 minutes
Water wash1 minute
Differentiator (mild acid) 1 minute
Water wash1 minute
Bluing1 minute
Water wash1 minute
95% ethanol1 minute
Eosin1 minute
95% ethanol1 minute
100% ethanol1 minute
100% ethanol1 minute
Xylene1 minute
Xylene2 minutes
Xylene 2 minutes
Coverslip