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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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The light coloration and appearance of droplets indicates excess water in the xylenes.
Note the lack of eosin in the sample.

Water in Xylene

While staining, water can get into the alcohols (following eosin) due to carryover between steps. When reagents are not changed regularly, the water content will continue to increase and will be transferred to the xylenes that follow before coverslipping. This excess water in the xylene can, over time, cause seeping of the eosin from the tissue. It is seen on the slide as a pink haze. This artifact can occur even if the xylene is not visibly contaminated with water.
Carryover is not the only means of getting water into the xylenes. Ambient humidity can cause changes in how quickly the xylene becomes contaminated with water.