Routine Mayer's Hematoxylin and Eosin Stain (H&E)

Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology (Third Edition). American Registry of Pathology ( Luna, Lee G., HT(ASCP) (editor)), McGraw Hill Publishers, New York 1960 (Progressive Stain)

Summary
Mayer's hematoxylin is used because it eliminates the necessity for differentiation and bluing of the section. It can be considered a progressive stain which produces a stained section with a clearly defined nuclei while the background is completely colorless.
The biggest objection to Mayer's hematoxylin as used in the past, has been that stained slides often fade after 1 to 3 years. This problem can be eliminated, however, when the slides are washed, after the hematoxylin, in running water for a minimum of 20 minutes.
This method gives consistent results even when more than one person stains sections from the same block. Also, slides may be left in the hematoxylin for hours with-out overstaining. Because of the simplicity of the technique, it is possible to teach others to use it within a shorter time as well as a definite reduction in time performance of the stain itself.
Fixation
Any well fixed tissue.
Technique
Paraffin, celloidin, or frozen
Solutions
  • Mayer's Hematoxylin
  • Eosin Solutions
  • Gram's or Lugol's Iodine
Staining Procedure
  1. Deparaffinize and hydrate to water
  2. If sections are Zenker-fixed, remove the mercuric chloride crystals with iodine and clear with sodium thiosulphate (hypo)
  3. Mayer's hematoxylin for 15 minutes
  4. Wash in running tap water for 20 minutes
  5. Counterstain with eosin from 15 seconds to 2 minutes depending on the age of the eosin, and the depth of the counterstain desired. For even staining results dip slides several times before allowing them to set in the eosin for the desired time
  6. Dehydrate in 95% and absolute alcohols, two changes of 2 minutes each or until excess eosin is removed. Check under microscope
  7. Clear in xylene, two changes of 2 minutes each
  8. Mount in Permount or Histoclad
Results
Nuclei - blue - with some metachromasia
Cytoplasm - various shades of pink-identifying different tissue components
Remarks
The adhesives used to attach sections onto the slides (gelatin, egg albumen) will sometimes stain, in areas around the section, with Mayer's hematoxylin. This will give the slides a slightly dark appearance but in no way affects the nuclear staining. To remedy this, use 10-12% glacial acetic acid in 95% alcohol, to "clean" the slides after Mayer's hematoxylin. Following with a few dips in saturated aqueous lithium carbonate, the nuclei will blue immediately. This is optional, for the 20-minute wash in running water is sufficient to blue the nuclei. This step will in no way alter or minimize the staining of the nuclei.
Reference
Histopathology Laboratories, Armed Forces Institute of Pathology, Washington, D.C. 20305.