Hematoxylin, Dehydration, & Mounting

Hematoxylin: 30 seconds–10 minutes; depends on concentration and pathologist preference

Tech Tip: The main difference between the 2 most common types of hematoxylin is concentration: Mayer’s = 1gm/L and Gill’s = 2gm/L.

If the hematoxylin incubation time is too long or too short, then the following may occur:
Dissatisfied pathologist difficulty distinguishing cellular morphology.

If the hematoxylin is exposed to light, then the following may occur:
Dissatisfied pathologist oxidation creating crystalline precipitates that are observed under the microscope; these crystalline structures can be distracting and, in some instances, make accurate diagnosis difficult.

Dehydration and Mounting:

  1. Dehydration: graded alcohol (3–10 minutes) and xylene or xylene substitute (5–15 minutes)
  2. Mounting Media: non-aqueous (permanent chromogens) vs. aqueous (non-permanent chromogens)

Tech Tip: Non-permanent chromogens like AEC are soluble in alcohols. It is important to air-dry these slides and use an aqueous mounting media.

No or inadequate dehydration can result in:
Cloudy appearance water is not fully removed creating a hazy appearance to the tissue; cellular morphology can also be affected.

Use of the wrong dehydration method or inappropriate mounting media can result in:
Weak staining non-permanent chromogens can fade if used with alcohols or with non-aqueous mounting mediums.