Hematoxylin Eosin Staining: Difference between revisions
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''' | '''Hematoxylin-eosin staining''' (HE) is a transparent staining used for the preparation of semi-thin sections. | ||
== | == Preparing solutions == | ||
=== Hematoxylin === | === Hematoxylin === | ||
* | * hematoxylin is supplied to the laboratory as a yellowish crystalline powder; | ||
* | * in order for the hematoxylin solution to stain, it must be oxidised with oxidising agents (e.g. sodium or potassium iodate, ferrous ions). This converts it to hematein; | ||
* | * the hematein must also be made into a coloured so-called hematein varnish using a mordant; | ||
* | * the most common mordants include alum (e.g. potassium alum = potassium aluminium sulphate), according to which we distinguish types of haematoxylins; | ||
* | * among the frequently used haematoxylins we mention e.g. Harris', Gill's, Heidenhein's | ||
=== Eosin === | === Eosin === | ||
There are several types of eosin. They differ in solubility, chemical composition and also in colour: | |||
# ''' | # '''bromeosins''' – eosin yellow, red | ||
# ''' | # '''iodeosines''' – erytrosins | ||
== | == Staining == | ||
[[ | [[File:Smooth muscle cells; stained with hematoxylin-eosin..jpg|thumb|Smooth muscle cells stained with hematoxylin-eosin]] | ||
The tissue section on the slides are ready for staining, which consists of the following steps: | |||
* | * deparaffinization ; | ||
* | * hydration (washing in tap water); | ||
* | * staining; | ||
* | * dehydration; | ||
* | * clearing; | ||
* | * mounting; | ||
=== | === Deparaffinization and hydration === | ||
The cuts must be free of paraffin before staining in aqueous solutions. This is done in a descending '''series of graded alcohols'''. | |||
; | ;Procedure | ||
{| class="wikitable" | {| class="wikitable" | ||
|- | |- | ||
| 1 || | | 1 || xylene || '''5 min''' | ||
|- | |- | ||
| 2|| | | 2|| xylene || '''5 min''' | ||
|- | |- | ||
| 3 || 96 % | | 3 || 96 % ethanol || '''3–5 min''' | ||
|- | |- | ||
| 4 || 80 % | | 4 || 80 % ethanol || '''3–5 min''' | ||
|- | |- | ||
| 4 || 70 % | | 4 || 70 % ethanol || '''3–5 min''' | ||
|- | |- | ||
| 5 || | | 5 || tap water|| '''5 min''' | ||
|} | |} | ||
=== | === Staining === | ||
# | # deparaffinized and hydrated sections are first stained with hematoxylin solution for '''3-10 minutes'''. The staining time depends on: | ||
#* | #* type of hematoxylin; | ||
#* | #* age of the solution; | ||
#* | #* type of tissue; | ||
#* | #* fixation. | ||
# | # washing in running water | ||
# | # a possible step is the so-called differentiation in acid ethanol, briefly and under the control of a microscope | ||
# | # washing in running water '''5 minutes''' | ||
# | # followed by eosin staining '''1–3 minutes''' | ||
# | # rinse in distilled water | ||
<small> | <small>If the cuts are recoloured, we briefly differentiate in 80% alcohol.</small> | ||
=== | === Dehydration === | ||
Drainage is carried out in '''ascending series of graded alcohol (reverse descending)'''. | |||
* | * xylene 5 minutes, | ||
* | * xylene 5 minutes. | ||
At the same time, the sections become '''clear''' in the xylene. After clearing, they must be completely transparent, no whitish cloudy areas must remain, which is a sign that the sections have not been well dehydrated. | |||
=== | === Mounting of stained sections === | ||
The mounting medium must be a transparent substance with a high refractive index (as close to glass as possible) that will not discolour the tissue. | |||
==== | ==== Types of mounting media ==== | ||
; | ;Not mixable with water | ||
Soluble in xylene. The sections must be properly drained and saturated with e.g. xylene. | |||
* | * Ex: formerly Canadian balsam, cedar oil, today e.g. Solakryl. | ||
; | ;Mixable with water | ||
Water soluble, closing without prior dehydration. | |||
* | * Ex.: glycerine, glycerine gelatin, levulose syrup | ||
==== | ==== Method of execution ==== | ||
# | # A drop of mounting medium is dropped onto the slide, the cover slip is carefully applied from an oblique angle to avoid air bubbles. Any bubbles can be squeezed out by applying slight pressure to the coverslip, e.g. with tweezers, etc. | ||
# | # The cut must not dry out during mounting. | ||
# | # The finished slide is placed in a thermostat (37 °C) where the mounting medium dries. | ||
<noinclude> | <noinclude> | ||
== Links == | == Links == | ||
=== Related articles === | === Related articles === | ||
* [[Basic staining methods]] | * [[Basic Staining Methods|Basic staining methods]] | ||
* [[Histochemistry]] | * [[Histochemistry]] | ||
* [[Burri staining]] | * [[Burri dyeing|Burri staining]] | ||
* [[Gram staining]] | * [[Gram staining]] | ||
* [[Chromosome staining]] | * [[Chromosome staining]] | ||
| Line 105: | Line 105: | ||
[[Category:Histology]] | [[Category:Histology]] | ||
[[Category: | [[Category:Pathology]] | ||
Latest revision as of 20:51, 10 November 2023
Hematoxylin-eosin staining (HE) is a transparent staining used for the preparation of semi-thin sections.
Preparing solutions[edit | edit source]
Hematoxylin[edit | edit source]
- hematoxylin is supplied to the laboratory as a yellowish crystalline powder;
- in order for the hematoxylin solution to stain, it must be oxidised with oxidising agents (e.g. sodium or potassium iodate, ferrous ions). This converts it to hematein;
- the hematein must also be made into a coloured so-called hematein varnish using a mordant;
- the most common mordants include alum (e.g. potassium alum = potassium aluminium sulphate), according to which we distinguish types of haematoxylins;
- among the frequently used haematoxylins we mention e.g. Harris', Gill's, Heidenhein's
Eosin[edit | edit source]
There are several types of eosin. They differ in solubility, chemical composition and also in colour:
- bromeosins – eosin yellow, red
- iodeosines – erytrosins
Staining[edit | edit source]
The tissue section on the slides are ready for staining, which consists of the following steps:
- deparaffinization ;
- hydration (washing in tap water);
- staining;
- dehydration;
- clearing;
- mounting;
Deparaffinization and hydration[edit | edit source]
The cuts must be free of paraffin before staining in aqueous solutions. This is done in a descending series of graded alcohols.
- Procedure
| 1 | xylene | 5 min |
| 2 | xylene | 5 min |
| 3 | 96 % ethanol | 3–5 min |
| 4 | 80 % ethanol | 3–5 min |
| 4 | 70 % ethanol | 3–5 min |
| 5 | tap water | 5 min |
Staining[edit | edit source]
- deparaffinized and hydrated sections are first stained with hematoxylin solution for 3-10 minutes. The staining time depends on:
- type of hematoxylin;
- age of the solution;
- type of tissue;
- fixation.
- washing in running water
- a possible step is the so-called differentiation in acid ethanol, briefly and under the control of a microscope
- washing in running water 5 minutes
- followed by eosin staining 1–3 minutes
- rinse in distilled water
If the cuts are recoloured, we briefly differentiate in 80% alcohol.
Dehydration[edit | edit source]
Drainage is carried out in ascending series of graded alcohol (reverse descending).
- xylene 5 minutes,
- xylene 5 minutes.
At the same time, the sections become clear in the xylene. After clearing, they must be completely transparent, no whitish cloudy areas must remain, which is a sign that the sections have not been well dehydrated.
Mounting of stained sections[edit | edit source]
The mounting medium must be a transparent substance with a high refractive index (as close to glass as possible) that will not discolour the tissue.
Types of mounting media[edit | edit source]
- Not mixable with water
Soluble in xylene. The sections must be properly drained and saturated with e.g. xylene.
- Ex: formerly Canadian balsam, cedar oil, today e.g. Solakryl.
- Mixable with water
Water soluble, closing without prior dehydration.
- Ex.: glycerine, glycerine gelatin, levulose syrup
Method of execution[edit | edit source]
- A drop of mounting medium is dropped onto the slide, the cover slip is carefully applied from an oblique angle to avoid air bubbles. Any bubbles can be squeezed out by applying slight pressure to the coverslip, e.g. with tweezers, etc.
- The cut must not dry out during mounting.
- The finished slide is placed in a thermostat (37 °C) where the mounting medium dries.
Links[edit | edit source]
Related articles[edit | edit source]
Literature[edit | edit source]
- VACEK, Zdeněk. Histologie a histologická technika. Díl 2, Histologická technika. 1. edition. Institut pro další vzdělávání pracovníků ve zdravotnictví, 1996. 184 pp. ISBN 80-7013-202-7.
