SPI-Chem Stains for Light Microscopy
A simple outline of H&E (hematoxilin and eosin) procedures
We are sometimes asked either by students or those beginning in the field
for an over view of "H & E procedures". While there are obviously a
wide assortment of published texts on the
subject, not everyone wants to rush out and purchase an expensive book
and furthermore, sometimes one needs only a general "feel" for what H&E
really means.
We find
one of the best references for H & E techniques is on the website of
the University of Bristol.
According to the University of Bristol information, the "general" H&E
approach is the following:
1] Dewax sections, rinse in alcohol, rinse in water.
2] Stain with Harris' haematoxylin - 10 minutes.
3] Wash and blue in running tap water - 1 minute.
4] Differentiate in acid alcohol (1% hydrochloric acid in 70% alcohol) - 10
seconds.
5] Wash and blue in running tap water - 5 minutes.
6] Stain with eosin - 4 minutes.
7] Wash in tap water.
8] Dehydrate, clear and mount.
At the end of the procedure, one can expect to see the following in the way
of results:
- Nuclei - blue.
- Other tissue components - shades of red and pink.
To make up the "Harris haematoxylin", use the following recipe of
ingredients:
a) Haematoxylin - 5g.
b) 100% alcohol - 50mls
c) Potassium alum - 100g.
d) Distilled water - 1 litre
e) Mercuric oxide - 2.5g.
f) Glacial acetic acid - 40mls
Preparation procedure for the "Harris haematoxylin"
1] Dissolve the potassium alum in the water by warming and stirring.
2] Dissolve the haematoxylin in the alcohol and add.
3] Bring rapidly to the boil remove from heat and add the mercuric oxide.
4] Cool, add the acetic acid and filter.
5] Ready for use immediately.
Preparation of eosin:
1] Prepare 1% eosin Y
(yellowish) in TAP water.
2] Add a crystal of thymol to prevent the growth of moulds.
3] Now ready to use in the H & E procedure described above.
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Wednesday March 10, 2010
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