Understanding Hematoxylin & Eosin Staining Part 1 – Dye, Counterstain & Quality Assurance

by | Jul 1, 2021 | LabStore Highlights | 0 comments

For centuries scientists and researchers have used tissue specific dyes to advance the knowledge and comprehension of cells and organs. After developing methods of preserving tissues, then performing thin layer sectioning, specimens then had to be stained in such a way as to show the morphology and variations of tissue types. Additionally, this would give the scientist the ability to visualize normal v/s abnormal tissue, and thus lead to diagnostic pathology.

The Hematoxylin & Eosin (H&E) stain is the most common and universally known histology staining technique and is based on the study of nuclear v/s cytoplasmic staining in tissue. Specific dyes have an affinity for specific molecules in tissues. The concept of ‘acidophilic’ dyes (having a pH of ˂ 7), and ‘basophilic’ dyes (having a pH of ˃ 7), is the foundation of a dye’s specificity for tissue elements. The nucleus of a cell, which is the primary point of interest in pathology, has negatively charged molecules and will have a strong affinity for positively charged molecules in certain dyes.

Natural State & Oxidized State

Hematoxylin is a natural dye extract which is formed in the Logwood tree found in Mexico and Central America.  To date there has been no other substance or source found to provide the quality and staining morphology better than hematoxylin.  Hematoxylin in itself has little to no staining capacity.  When oxidized it forms a substance called ‘hematein’.  The chemical compound images illustrate this process.  Hematein, then bound to aluminum, forms an overall positive charge.  The chromatin material found in the nucleus of cells is strongly negative in its charge.  So now you can see the strong affinity of a positively charged hematein/aluminum compound to a negatively charged nuclear chromatin.  This gives us the richness and depth of the hematoxylin dye. 

Hematoxylin comes in various forms and chemistries such as Harris’, Mayer’s, Gill’s, and others. Some are stronger and more desirable for tissue, while others are less intense and used for cytologic staining. Hematoxylin can be placed into two categories: Progressive and Regressive. Because the stronger hematoxylins have a tendency to overstain tissue, and the excess stain must be removed by differentiation, these hematoxylins require a ‘regressive’ method of staining. This is where hematoxylin stain is applied, then differentiated out with a mild acid alcohol solution, then the counter stain is applied. A controlled differentiation step will pull out most of the stain leaving intense staining in the chromatin (DNA) material of the nuclei. With the stronger hematoxylin there is the unfortunate incident of non-specific staining in acid mucins and proteoglycans. The images show residual hematoxylin staining in the goblet cells of the small intestine. On the left you see the light blue mucinous staining in the lumen cavity and cells. On the right you can see a superior differentiation applied where the excess hematoxylin has been removed. The stain has been regressed. Progressive hematoxylins typically do not require a regressive step in the procedure. The chemistries of these hematoxylins are different and leave little to no residual staining, but they do not stain with the same intensity of regressives.

Hematoxylin gives a natural staining response of reddish-purple and after staining must be treated with an alkaline rinse to bring out the deep bluish-purple color. This post staining step is called ‘bluing’. The pH of this rinse is typically 7.6 – 9.0. With the progressive hematoxylins oftentimes tap water can be used for this step because it is on the average about 7.5 pH, however, tap water is very inconsistent from region to region based on the local water treatment methods. It is not recommended for regressive hematoxylins.

As mentioned in the beginning of this article, the hematoxylin stain is a basophilic dye with an affinity for negatively charged molecules. It has an overall positive charge, but when it becomes diluted from repeated staining steps (either by water or depleted dye content), the stain will begin to rise in pH and thus lose its affinity for nuclear chromatin. Slide staining quality should always be monitored when doing multiple staining runs through the same hematoxylin solution. Staining methods should be developed and validated based on, among other things, the staining capacity of the hematoxylin.

In this article we have given you some basic but essential information on hematoxylin that will assist you producing distinct, crisp nuclear detail in your staining. In part 2 we will discuss Eosins and the balance of coloration between nuclear and cytoplasmic staining.

Brown, S., “The Science & Application of H&E Staining”, Workshop in Technical Develop Series, Lab Management Consultants, 2011.
Brown, S., “A Systems Approach to H&E Staining”, Workshop presentation, National Society for Histotechnology Symposium, 2013.
Hansen, S., Images in H&E Staining, LBS, 2011.


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