Hematoxylin-eosin staining Principle, Procedure, protocol (H&E stain)

hematoxylin and eosin stain is commonly used stains in the histopathology and cytology section of the clinical laboratory. It is preferred for viewing cellular and tissue structure detail by pathologists.

It demonstrates the broad range of cytoplasmic, nuclear, and extracellular matrix features of the tissue.

Hematoxylin eosin stain Principle

First of all, wax cleared tissues are rehydrated which helps in the entry of dyes. Then tissues are subsequently exposed to the basic dye Hematoxylin and an acidic dye Eosin. These dyes stain the acidic and the basic components of the tissue.

What are thehematoxylin eosin (H & E) stain reagents ?

Following reagents are used in H & E stain.

1. Harris Hematoxylin reagent
2. Acid Alcohol
3. Ammonia water
4. Alcohol with eosin solution
5. Other reagents, Xylol, absolute alcohol, rectified spirit, methylated spirit.

Hematoxylin eosin stain procedure

H & E stain has the following steps in the procedure.

1. Place the section fixed tissue slid in the xylene for 3 minutes.
2. In the next step, transfer it to the absolute alcohol for 3 minutes.
3. Place it in the methylated spirit for two to three minutes.
4. In this step, wash the slide in running water and place the slid in a Hematoxylin jar for three to five minutes.
5. Wash in clear water. Remove extra dye with 1% acid alcohol.
6. In this step, two to three dips the section in ammonia water until it becomes blue in color.
7. Wash in running water.
8. Counterstain with eosin for two minutes.
9. Wash in tap water for 20 seconds.
10. Dehydrate the section by placing it gradually increasing concentration of alcohol 70%, 95%, and absolute alcohol.
11. In the last step, Clear with xylene and mount with Canada balsam.

Hematoxylin eosin stain result

1. Muscle and keratin: Bright pink
2. Nucleus Bright blue
3. Collagen: Pink
4. Red Blood Cells: Orange Red

Difference between h & e stain

Hematoxylin is a basic dye, it stains the acidic component of tissue, while eosin is an acidic dye it colors the basic components of the tissue.

How does H & E stains work?

Hematoxylin is a basic dye while eosin is an acidic dye. when wax cleared tissue section is placed in the acidic dye (hematoxylin) it binds with basic components of the tissue but doesn’t color the acidic parts of the tissue.

Same as, when this section is placed in the acidic dye, it colors the basic parts of the tissue without interfering with the acid parts of the tissue.

How to prepare hematoxylin stain

First, dissolve the hematoxyline separately in alcohol and in potassium alum in water. Mix and boil. Remove from flame and add mercuric oxide. Rehear for a minute until its color change to purple.

Remove from flame and cool in the basin of cold water. After the cooling stain is ready for use. Sometimes glacial acetic acid is added to improve the stain quality.