The H&E Stain: Far From Routine Part 2

This blog is a followup to the previous article “The H&E Stain: Far From Routine”.  In that article, the basics of the routine hematoxylin and eosin (H&E) stain were discussed.  Now we shall discuss how to trouble shoot the routine H&E and how to ensure a high quality stain, once you have worked with your pathologist to determine the best stain result for your laboratory.

A standard H&E staining protocol is provided below.  It applies to either a manual or automated staining procedure.  While it may not be ideal for your laboratory, it can be used as a starting point.  The final color rendition of your H&E stain should be determined by working with your pathologists.  This will consequently make their job easier.  Each day, once the H&E stain set up is completed, you should run down one test slide to confirm that the staining is optimal.  This also will help you to document quality control procedures.

Hematoxylin and Eosin (H&E) Stain

PRINCIPLE: Hematoxylin stains nuclear material a dark blue, while the eosin stains cytoplasm and connective tissue varying shades of pink.


  1. Harris’ hematoxylin
  2. Alcoholic Eosin
  3. Clarifier (i.e. acid alcohol)
  4. Bluing reagent (i.e. ammonium hydroxide)


  1. Cut paraffin sections 4-5 microns.  Bake at 60 C for 45 minutes; allow to cool.
xylene2 x 5 minRemoves paraffin; this is a minimum time
100% alcohol2 x 2 minRemoves xylene
95% alcohol1 x 2 minBegins hydration
Running water2 minHydrates the sections
Harris’ Hematoxylin5 minStains nuclei and tissue
Running water2 minRemoves excess hematoxylin
Clarifier1 minRemoves hematoxylin from tissue
Running water2 minStops clarifier action
Bluing reagent1 minChanges hematoxylin from red to blue with positive ions
Running water2 minStops bluing action
95% alcohol1 minReadies for eosin
Eosin1 minStains cytoplasm pink
95% alcohol15 secondsDifferentiates eosin into 3 shades
100% alcohol3 x 2 minDehydrates
Xylene2 x 5 minReadies for coverslip

Each step in the H&E stain procedure is essential, and if not managed correctly, any of them can cause suboptimal slides.  We shall begin discussion here and continue through the next blog, identifying potential problems and solutions along the way.

  1. Deparaffinization and dehydration. Many H&E and special stain protocols begin with step #1 as simply “Deparaffinize and hydrate to water”. In fact, nothing could be more complicated.  The sections to be stained are surrounded by a matrix of paraffin wax, which is insoluble in water.  All of the paraffin must be removed to ensure optimal staining.  Two changes of xylene (or xylene substitute) for 5 minutes each may accomplish this in a low volume laboratory; however a high volume laboratory may need to use three changes of xylene / xylene substitute to completely remove all paraffin.  In automated H&E stainers, it is important to adjust the height of xylene (and all reagent) containers to ensure that the sections next to the label end of the slide are being deparaffinized sufficiently.
  1. Harris’ hematoxylin is recommended for histology. Gill’s hematoxylin is generally used for cytology, and as a counterstain in many special stains. Harris’ hematoxylin continues to oxidize as it sits in the original container, and in the stain containers; hence, it should be filtered through Whatman #1 filter paper prior to use for the days’ staining runs.  This will prevent precipitate from adhering to the microscope slides.
  1. Clarifier. Harris’ hematoxylin is a regressive stain.  A clarifier solution must be used to remove excess hematoxylin staining from both the nuclei and other tissue elements.  The original formulation  is 1% hydrochloric acid in 70% ethanol, for “a few quick dips”.  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.  The recommended starting point for clarifier on an automated stainer is to use the following for 30 seconds:

100% ethanol                            700 ml

tap water                                  2600 ml

hydrochloric acid (concentrated)  2.0 ml

Once made, the resulting intensity of the nuclear stain can be adjusted by changing the time of the clarifier incubation.  Alternatively, one may adjust the amount of acid added to the alcohol (i.e. decrease from 2.0 ml to 1.5 ml).  These adjustments will allow you to obtain a final stain with the required nuclear staining intensity.

  1. Bluing reagent. The recommended starting point for bluing reagent is:

Tap water                                                    3500 ml

Ammonium hydroxide (concentrated)               1.5 ml

As with the clarifier solution, results can be obtained by adjusting the time of incubation and/or concentration of the solution.  Alternatively, one can use lithium carbonate as a bluing reagent, or simply use a running water step to “blue” the nuclei.

  1. Eosin.  Working eosin solution is the most stable reagent of the H&E stain.  Hundreds of slides can be stained with the same batch of eosin on board an automated stainer.  The incubation time is usually kept short, as eosin penetrates and stains rapidly and reproducibly (i.e. 1-2 minutes).  The variable step in eosin staining is the subsequent 95% alcohol incubation, which produces the final “three shades of pink” within the tissue sections.  The number of 95% stations (i.e. one or two), and the incubation times (i.e. 30 seconds to 2 minutes) will determine the final quality of eosin staining.  Finally, it is important that the final 100% alcohol stations after the eosin staining remain uncontaminated with water.  Any water left in the sections after the coverslip is applied may cause the eosin to “bleed out” of the section.

In summary, the information contained herein will hopefully help you to manage and maintain consistent high quality of the H&E slides produced in your laboratory.


  1. Theory and Practice of Histological Techniques. JD Bancroft, A Stevens ed. Churchill Livingstone, NY.  Fourth edition. 1996
  1. Theory and Practice of Histotechnology.  DC Sheehan, BB Hrapchak.  CV Mosby Company, St. Louis. First edition. 1980.
  2. Luna L.  AFIP. Manual of Histologic Staining Methods.  Third Edition. McGraw-Hill.

p39. 1968.  As modified by CM Chapman

  1. Dermatopathology Laboratory Techniques.  CM Chapman, I Dimenstein.  In press.
  2. The H&E Stain: Far from Routine.  CM Chapman.  Advance for Laboratory Professionals.  April 8, 2002.  Vol. 14 No.8