Staining Procedures

Most dyes used to visualize the membranes and organelles of the cell are water soluble. The embedded wax must therefore be removed prior to staining. This is done by effectively reversing the tissue processing schedule.

There are literally thousands of staining protocols and procedures in use. As an example, one of the most common stains, the Hematoxylin-Eosin stain, is presented below. For a detailed list of stain procedures we recommend that you visit the University of Bristol web site.

Hematoxylin and Eosin Stain


Hematoxylin is a natural compound extracted from a species of tree found in Mexico and the West Indies. The extracted compound is then oxidized to produce hematein, which is the active staining component of the hematoxylin stain. Hematoxylin stains must therefore be 'ripened' by oxidation before they can be used. Hematoxylin staining requires the use of a mordant (most commonly aluminum salts) and stains the nuclear components of cells a dark blue. Hematoxylin is used in combination with eosin because eosin stains the cytoplasmic organelles varying shades of pink, red or orange. The combination of the two stains provides a broad range of morphological information about the section.

H & E Stain with Harris' Hematoxylin


STAINING PROCEDURE

  1. Stain rehydrated sections in Hematoxylin solution for 20-40 minutes.
  2. Wash in tap water for 1-5 minutes, until sections turn blue ("bluing").
  3. Differentiate sections in 70% ethanol—containing 1% HCl—for 5 seconds. This removes excess dye, allowing nuclear details to emerge.
  4. Wash 1-5 minutes in tap water until blue.
  5. Stain in Eosin solution for 10 minutes.
  6. Wash 1-5 minutes in tap water.
  7. Dehydrate, clear and mount.

Note the use of tap water in the washing steps—tap water provides the alkanlinity necessary for the "bluing" process.


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