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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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Poor deparaffinization leaves wax in the tissue, which will impede the staining ability
of the dyes. Note the area indicated by the arrow. This area shows how the wax
that was left behind during deparaffinization obscures the ability for the tissue to absorb
dyes, impairing diagnosis.

The light-colored areas on this sample indicate remaining paraffin. In this
high level image, several areas of inadequate deparaffinization can be seen.

Inadequate Deparaffinization

The use of clean and fresh dewaxing reagents is essential for the removal of paraffin from the slide prior to the addition of the dyes. While xylene is the most commonly used solvent, xylene substitutes are gaining in popularity because they are considered less hazardous and more ecofriendly. Water in solvents, whether from reagent contamination or a high humidity environment, reduces the ability of the solvent to remove the paraffin. Remaining paraffin prevents the dyes from penetrating the tissues, thus giving an uneven appearance.
The simplest way to prevent this from occurring is to change reagents more frequently. Adding a small of amount of desiccant pellets (about a tablespoon per reagent vessel) will also reduce water contamination within solvents. These measures are especially important when using a xylene substitute, as these reagents tend to be far less tolerant of any water contamination than xylene.