H&E staining procedure

https://www.leicabiosystems.com/en-fr/knowledge-pathway/he-
staining-overview-a-guide-to-best-practices/
• The dye itself is extracted from the tree Haematoxylum campechianum. Oxidation of
the hematoxylin produces hematein, which is the actual dye used in an H&E stain.
Addition of the mordant improves the ability of the hematein to attach to the anionic
(negatively charged) components of the tissues.
• Hematoxylins are typically classified by the mordant used before staining
• Mordants strengthen the positive ionic charge of the hematin. This aids the bonding of
the hematin to the anionic tissue component, which is most commonly chromatin. The
type of mordant also influences the final color of the stained components. The most
common mordant used in routine histology is aluminum ammonium sulfate (alum).
This mordant causes the nuclei to be red in color, which is then changed to the more
familiar blue color when the sample is later rinsed with a weakly basic solution.
• Adding small amounts of acetic acid to the hematoxylin periodically will aid in
maintaining appropriate pH and can extend the life of the stain.
• Mayer's hematoxylin is an alum hematoxylin
• For these applications, Mayer's is used to stain the nuclei and then
blued without the use of a differentiator. Mayer's is a water-based
stain.
• In the case of hematoxylin, hydrochloric acid (for rapid
differentiation) and acetic acid (for slower, more controlled
differentiation) are most commonly used.
• Harris hematoxylin is another commonly used alum hematoxylin
• One challenge when using Harris is that it is best differentiated with a
mild acid, as opposed to the more commonly used hydrochloric acid-
based differentiators.
• Harris is an alcohol-based stain.
• Eosin Y is the most commonly used form of eosin and may be used in
both water and alcohol.
• The addition of a small amount of acetic acid will also sharpen the
staining of the eosin.
• So for those who want to see richer looking reds, phloxine may be
added.
• Water is used as a differentiator for eosin. It is common to follow the
eosin step with 95% ethanol. The ethanol aids with rinsing the slide,
while water pulls excess eosin from the tissue.
 There are typically three types of H&E stains: progressive,
modified progressive, and regressive.
• Progressive staining occurs when the hematoxylin is added to the
tissue without being followed by a differentiator to remove excess
dye.
• Regressive staining occurs when the hematoxylin is added to the
tissue with being followed by a differentiator to remove excess dye.
• Simple regressive stain that provides a nice balance of nuclear and
cytoplasmic stains. This protocol is designed with a mild acid
differentiator
Remove the Wax
• Following the preparation of a paraffin section, all the elements are
infiltrated with and surrounded by paraffin wax which is hydrophobic
and impervious to aqueous reagents. The majority of cell and tissue
components have no natural color and are not visible. The first step in
performing an H&E stain is to dissolve all the wax away with xylene
(a hydrocarbon solvent).
Hydrate the Section
• After thorough de-waxing, the slide is passed through several changes
of alcohol to remove the xylene, then thoroughly rinsed in water. The
section is now hydrated so that aqueous reagents will readily penetrate
the cells and tissue elements.
Apply the Hematoxylin Nuclear Stain
• The slide is now stained with a nuclear stain such as Harris
hematoxylin, which consists of a dye (oxidized hematoxylin or
hematein) and a mordant or binding agent (an aluminum salt) in the
solution. Initially this stains the nuclei and some other elements a
reddish-purple color.
Complete the Nuclear Stain by “Blueing”
• After rinsing in tap water, the section is “blued” by treatment with a weakly
alkaline solution. This step converts the hematoxylin to a dark blue color. The
section can now be rinsed and checked to see if the nuclei are properly stained,
showing adequate contrast and to assess the level of background stain.
• Scott's tap water substitute
sodium hydrogen carbonate --- 10 gm
magnesium sulphate ----------- 100 gm
distilled water ------------------- 5 L
• 0.2% Ammonia Water Solution (Bluing):
Ammonium hydroxide (concentrated) ----- 2 ml
Distilled water ------------------------------- 1000 ml
Mix well.
Remove Excess Background Stain
(Differentiate)
• On most occasions when Harris hematoxylin is employed, a
differentiation (destaining) step is required to remove non-specific
background staining and to improve contrast. A weak acid alcohol is
used. After this treatment, blueing and thorough rinsing is again
required. Staining methods that include a destaining or differentiation
step are referred to as “regressive” stains
• 1% Acid Alcohol Solution (for differentiation):
Hydrochloric acid --------------- 1 ml
70% ethanol --------------------- 100 ml
Mix well.
Apply the Eosin Counterstain
• The section is now stained with an aqueous or alcoholic solution of
eosin (depending on personal preference). This colors many
nonnuclear elements in different shades of pink.
Rinse, Dehydrate, Clear and Mount (Apply
Cover Glass)
• Following the eosin stain, the slide is passed through several changes
of alcohol to remove all traces of water, then rinsed in several baths of
xylene which “clears” the tissue and renders it completely transparent.
A thin layer of polystyrene mountant is applied, followed by a glass
coverslip. If the stain and all the subsequent steps have been properly
performed, the slide will reveal all the important microscopic
components and be stable for many years.
Procedure
1. Deparaffinization: flame the slide on burner and place in the xylene. Repeat the treatment to remove the
wax.
2. Hydration: Drain xylene and hydrate the tissue section by passing through decreasing concentration of
alcohol baths (100%, 90%, 80%, 70%) and water.
3. Nuclear Staining: Stain in hematoxylin for 3-5 minutes.
4. Wash in running tap water until sections “blue” for 5 minutes or less.
5. Differentiation: selective removal of excess dye from the section). Dip in 1% acid alcohol (1% HCl in 70%
alcohol) for a few seconds.
6. Blueing: Rinse in running tap water. Dip in ammonia water until the sections become blue, followed by
tap water wash.
7. Counterstain: Stain in 1% Eosin Y for 10 minutes.
8. Wash in tap water for 1-5 minutes.
9. Dehydration: Dehydrate in increasing concentration of alcohols.
10.Clearing: Put slides in two xylene baths for clearing.
11.Mounting: Mount in DPX or other mounting media.
12.Observe under compund microscope.
 Xylene 2 minutes
Xylene 2 minutes
100% ethanol 2 minutes
100% ethanol 2 minutes
95% ethanol 2 minutes
Water wash 2 minutes
Hematoxylin 3 minutes
Water wash 1 minute
Differentiator (mild acid) 1 minute
Water wash 1 minute
Bluing 1 minute
Water wash 1 minute
95% ethanol 1 minute
Eosin 45 seconds
95% ethanol 1 minute
100% ethanol 1 minute
100% ethanol 1 minute
Xylene 2 minutes
Xylene 2 minutes
Coverslip
Requirements
1.Harri’s Hematoxylin stain
A = 1 gm hematoxylin in 10 ml ethanol
B = 20 gm ammonium alum in hot distilled water
Mix A & B, boil and add 0.5 gm of mercuric oxide and filter.
2.Eosin solution
Yellow eosin = 1 gm
Distilled water = 80 ml
Ethanol = 320 ml
Glacial Acetic Acid = 2 drops
3.0.5% HCl
4.Dilute ammonia water