H&E staining

Hematoxylin solution modified according to Gill III,

Mayer’s hemalum solution and eosin Y solution

Focussing your histology targets.

The trio for
H & E staining
Results in just two steps:
Hematoxylin solution modified acc. to Gill III
or Mayer’s hemalum solution for nuclear
staining and eosin solution for counterstaining

The best known and most widely used staining procedure,

hematoxylin-eosin staining (H&E), uses hematoxylin
solutions for nuclear staining and eosin solutions for cyto-
plasmic staining.

In the first step, the nuclei are stained with a hematoxylin

solution. The nuclei stain blue, dark violet to black.

The second step is counterstaining with a xanthene dye, e.g.

eosin Y, eosin B or erythrosin B. In this process cytoplasm,
collagen, keratin and erythrocytes stain red.

H&E staining is the standard staining method used in histo-

logy. It gives an overview of the structure of the tissue,
enabling differentiation of the structures being examined
as normal, inflamed or degeneratively changed, or patho-
logical.

It is often possible to make a diagnosis based on the results of

H&E staining, or it can be determined which special staining
techniques are still necessary for subtyping.

H&E staining is used for the following material:

Paraffin sections, frozen sections and clinico-cytological
specimens, e.g. sputum, urine, FNAB, effusions, lavages.
 Benefits of H&E staining for you:

• Outstanding results from the combination

• Coordinated components

• Ready-to-use reagents

• Easy to use

• Developed, produced and tested

acc. to DIN EN ISO 13485

H & E staining procedure

Staining protocol
1. Deparaffinize sections in the usual way
and rehydrate
2. Stain in Mayer’s hemalum solution 3 min
or hematoxylin solution mod. acc. to Gill III 3 min
3. Rinse in HCl solution 0.1 % 2 sec
4. Differentiate in running tap water 3-5 min
5. Stain in eosin Y solution 0.5 % aqueous, 3 min
eosin Y solution 0.1 % aqueous 3-5 min
or eosin B solution 0.1 % aqueous,
erythrosin B solution 0.1 % aqueous 3-5 min
6. Rinse in tap water 30 sec
7. Ascending alcohol series,
2 x Neo-Clear ® or xylene
8. Mount with Neo-Mount®
or Entellan® new
Technical note: Before use, approx. 0.2 ml of acetic acid (glacial acetic acid) 100 % anhydrous (Merck Cat. No. 1.00063)
can be added per 100 ml of eosin solution to intensify the red color.

Results
Nuclei dark blue

eosin Y eosin B erythrosine B

Cytoplasm red-orange red red
Collagen, elastin, erythrocytes yellow-orange red-orange red-orange
Hematoxylin solution
modified acc. to Gill III Liver tissue
Product benefits
stained with hematoxylin solution modified acc. to Gill III
• Selection of a dye concentration
that meets the requirements
(histology = Gill III, cytology = Gill II)
Principle • Ready-to-use solution
In all hematoxylin staining procedures positively • Manufactured with Certistain® dyes
charged metal-hematein (in this case: aluminum) • No filtration necessary
complexes bind in an acid milieu to the negatively • Short staining time
charged phosphate remains of the cell nucleus-DNA. • Stable solution in storage and in use
In the Gill solutions, therefore, hematoxylin is • Good red/blue differentiation
oxidized to hematein with sodium iodate. Compared • No direct contact with dye powder
with the mercury oxidation that was common
in the past, this oxidation takes place in a controlled
manner, is more efficient and less harmful to the
environment.

Mayer’s hemalum
solution Product benefits
Intestinal tissue stained with Mayer’s hemalum solution
• Selective nuclear staining
• Conventional staining solution
Principle • Convenient to use
Mayer’s hemalum is one of the traditional staining • No overstaining
solutions used for nuclear staining in histology. • Ready to use
In the hemalum solution hematoxylin forms a com- • Economical
plex compound with aluminum ions. Selective • Reproducible results
nuclear staining is achieved through the acidity of • Good stability
the staining solution and an excess of alum. • No direct contact with dye powder

Hematoxylin solution mod. acc. to Gill III

and Mayer’s hemalum solution
Due to the addition of aluminum salts, both solu-
tions belong to the same group of hematoxylin
solutions – the so-called hemalums. Hematoxylin
solution acc. to Gill III and Mayer’s hemalum
solution are used for nuclear staining in histology
and in immunohistochemistry.
Both solutions require roughly the same amount
of time for staining, differentiation and bluing.
Hematoxylin solution modified acc. to Gill does
not require filtering before use; Mayer’s hemalum
must be filtered.
Both solution are produced and tested according to
DIN EN ISO 13485.
Eosin Y solution
0.5 % aqueous Product benefits
• Ready to use
• Usable as a stock solution
• Very economical
Principle • Stable for a long time after opening
Eosin Y (C.I. 45380) is an acidic colorant that • Selected raw materials
mainly binds to proteins. Aqueous eosin Y solution • High level of reproducibility
is the most frequently used cytoplasmic stain in • Suitable for use with Merck’s hema-
histology. The balance between nuclear staining toxylin solutions
with hematoxylin and cytoplasmic staining with • Tested applications
eosin is a matter of personal taste and personal • No direct contact with dye powder
experience. The concentration of the ready-to-use
solutions makes it possible to quickly and easily
obtain the desired result, by diluting if necessary.
Tested applications for the staining solutions are
available on the Internet under
http://microscopy.merck.de, or on request. Liver tissue stained with eosin Y solution

Ordering information
Product Merck Cat. No. Pack size
Eosin Y solution 0.5 % aqueous 1.09844 1l
Eosin Y (C.I. 45380) Certistain® 1.15935 25 g, 100 g
Eosin B (C.I. 45400) Certistain® 1.15934 25 g, 100 g
Erythrosin B (C.I. 45430) Certistain® 1.15936 10 g, 25 g
Mayer’s hemalum solution 1.09249 500 ml, 1 l, 2.5 l
Hematoxylin solution 1.05174 500 ml, 1 l, 2.5 l
modified acc. to Gill III
Hematoxylin (monohydrate) 1.15938 25 g, 100 g
(C.I. 75290) Certistain®
Hematoxylin cryst. (C.I. 75290) 1.04302 25 g, 100 g
Entellan® new 1.07961 100 ml, 500 ml
Neo-Mount® 1.09016 500 ml
Neo-Clear® (xylene substitute) 1.09843 5 l, 4 x 5 l

All the products named are CE certified. Current directions for use can be found on the Internet at http://microscopy.merck.de.
In addition to the ready-to-use staining solutions, it is also possible to prepare staining solutions using the relevant solid dyes.
The instructions for this can also be found on the Internet.

Auxiliary reagents
Ethanol absolute GR for analysis 1.00983 1 l, 2.5 l, 5 l
ACS, ISO, Reag. Ph Eur
Ethanol denatured with 1.00974 1 l, 2.5 l
approx. 1 % ethylmethyl ketone GR for analysis
 W 287121
08/04

Further information:
Merck KGaA
64271 Darmstadt, Germany
Phone +49 (0) 6151/ 72-0
Fax +49 (0) 6151/ 72-33 80
E-mail: microscopy@merck.de
http:// microscopy.merck.de

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