What exactly is a routine “H&E”? And what makes it routine? The first question is easy. “H” stands for ”hematoxylin” and “E” stands for “eosin”. Both are dyes used to stain tissue sections in histology. However, the procedure for correctly applying this combination of stains to tissue sections is far from routine.
Since both dyes are water soluble, the first step is to completely remove the paraffin that is present in the tissue sections from the microscope slides. Soaking in xylene, or a xylene substitute, followed by 100% and 95% alcohol, allow the slides to then be immersed in running water, which hydrates the sections.
The next step is to place the slides in a solution of hematoxylin, a natural dye obtained from the logwood tree Haematoxylon campechianum that will stain the nuclei of cells blue/black. The staining is enhanced by the addition of an aluminum, iron or lead salt, which acts as a mordant for the hematoxyin to bind to the tissue sites. This type of hematoxylin is referred to as “Harris” type, and is a “regressive’ stain. That is: the tissues are overstained with the hematoxylin, which stains all tissue elements, and then differentiated with an acid alcohol solution (i.e. “clarifier”) to remove excess hematoxylin, leaving only the nuclei of the cells stained.
The composition of the clarifier varies. The original procedure written by Lee Luna in the Armed Forces Institute of Pathology (AFIP) Manual specifies differentiation of the Harris hematoxylin solution with a “1% acid alcohol” solution for a time duration of “a few quick dips”. This solution is 1% hydrochloric acid in 70% ethanol. However, with the current use of automated stainers, “a few quick dips” needs to be quantified into seconds or minutes. This usually requires a dilution of the original clarifier solution, with some experiments to determine the exact time.
Harris’ hematoxylin can be compared to “Gill’s” or “Mayer’s” hematoxylin, which are used “progressively”. The longer the sections are left in these solutions, the darker the nuclei stain.
In either case, the resulting hematein-mordant stains nuclei a reddish color. Subsequent treatment in a weak basic aqueous solution, using either lithium carbonate or ammonium hydroxide, changes the dye molecules from red to blue; hence the term “bluing reagent”. The resulting blue color is dependent upon the freshness, type and age of the mordant. It is important that the pH of the bluing reagent is not too high, as section loss may occur during staining. Additionally, warm running tap water may be used as the “bluing reagent”, as it contains positive ions. The final blue color will be decided upon by the pathologist and histologist, who should agree what is optimal for the laboratory.
Eosin, the second dye in the H&E, is used to demonstrate the general histology of the tissue architecture. When used correctly, eosin should stain both cytoplasmic and tissue elements in three shades of pink. There are several types of eosin dye available, however Eosin Y is widely used for routine staining, as it is soluble in both water and alcohol. Usually a 0.5 % to 1.0 % solution of Eosin Y is made up in 80% alcohol, with a small amount of acetic acid added. Additionally, phloxine may be added to provide a more intense reddish color.
After staining in eosin for one to three minutes, the final shades of pink are determined by the differentiation steps following this staining. The differentiation can be carried out in running water, 95% or 100% alcohol. In any case the slides must be dehydrated completely after differentiation in several changes of 100% alcohol and xylene prior to coverslipping. Failure to do so may cause “bleeding” of the eosin dye in the final microscope slide and/or variable eosin staining.
Complete removal of paraffin is an essential first step in any H&E staining procedure. If xylene substitutes are being used, it may be necessary to adjust and lengthen times to guarantee paraffin removal. In addition, xylene substitutes do not tolerate traces of water, as xylene does. Thus, it is important also in the dehydration steps prior to coverslipping that the slides move through fresh changes of alcohol and xylene substitute. If any water remains in the sections during coverslipping, the final slides will appear cloudy. Additionally, the eosin stain may bleed out of the sections, as mentioned above.
This article has provided the basics of the H&E “routine” stain. Please keep an eye open for the next blog, which will provide additional information regarding how to maintain the quality of your H&E stain, once you have optimized it.
- Theory and Practice of Histological Techniques. JD Bancroft, A Stevens ed. Churchill Livingstone, NY. Fourth edition. 1996.
- Theory and Practice of Histotechnology. DC Sheehan, BB Hrapchak. CV Mosby Company, St. Louis. First edition. 1980.
- Luna L. AFIP. Manual of Histologic Staining Methods. Third Edition. McGraw-Hill.
p39. 1968. As modified by CM Chapman.
- Dermatopathology Laboratory Techniques. CM Chapman, I Dimenstein. In press.
- The H&E Stain: Far from Routine. CM Chapman. Advance for Laboratory Professionals. April 8, 2002. Vol. 14 No.8.
Questions and comments may be directed to:
Clifford M. Chapman, MS, HTL(ASCP), QIHC
Bioscience Solutions Group, LLC