Hematoxylin and Eosin Staining Procedure
We all know staining procedures change laboratory to laboratory and age of the stains . also there lot of short cut methods. But main staining principle & procedure should be same. So in this article I’m going to show you, Hematooxylin and Eosin procedure which I learn from my university under the supervision of histopathologist.
When we consider principle of Hematoxylin and Eosin staining procedure , we can divide it into 10 steps
1 De- Parafinization
2 Remove of syline
3 Rehydration
4 Nuclear staining
5 Differentiation
6 Bluing
7 Counter staining
8 Differentiation of eosin
9 Dehydration &
10 Clearing
1 De- Parafinization
When we consider step number 1 , de-parafinization , the main aim of this step is remove remaining paraffin wax from tissues. For that usually we use xylene reagent.
Dip in ,
Xyline I – 7 minutes &
Xyline II – 7 minutes
2 Remove of xylene
In this stage main is , remove xylene & replace xylene from Alcohol.
Ab.Alcohol I – 2 minutes
Ab.Alcohole II – 2 minutes
3 Re – Hydration
Next stage is re hydration , the main aim of these steps are remove alcohol from tissue & replace alcohol from water. Because stains are always water soluble , so for staining tissues should take in to water.
90% alcohol – 2 min
80% alcohol – 2 min
70% alcohol – 2 min
60% alcohol – 2 min
Finally all tissues take in to the water.
4 Nuclear Staining
Harris hematoxylin is used as nuclear staining dye .
Dip the slide in filtered Harris haematoxylin solution for 8 minutes.
Haematoxylin is a basic dye , so it can bind & stain acidic components of the cell.
Next wash the slide in running tap water for 5 minutes. Remember that it is necessary to use running water to remove unnecessary stain.
5 Differentiation
For differentiation we use , 2 quick dips in 1% acid alcohol
Then wash in running tap water for 3 minutes.
Since the acid solution alters the colour of the tissue to red , this step important to identify correct end-point & removes the excess staining.
You requires some practical experience, to check under microscope to assess the differentiation.
6 Bluing
Then perform the blueing step by dipping in 0.2% ammonia water , saturated lithium carbonate or Scott’s tap water for 2min
Then Immediately wash in running tap water for 3 min to stop blueing process.
This bluing process important to give , cool bluish purple color to the acidic cellular components.
Rinse in 95% alcohol to prepare tissue section for counter staining
7 Counter Staining
Next perform the counter stain by dipping slide in 1% eosin for 5 minutes.
After that should wash in running tap water 3 minutes.
This counter staining step important to , stain cytoplasm, muscle fibres, etc. This pink counterstain also helps to differentiate between nuclei and non-nuclear components in cells.
8 Differentiation of eosin
In this stage , Remove unnecessary eosin by using 95% alcohol
Quick 2 dips in 95% alcohol enough
9 Dehydration
In this step , we again remove water in histological sections.
For that we use dip in ascending concentration alcohol serious .
Alcohol replace water in tissues ,
70% alcohol – 2 dips
80% alcohol – 2 dips
95% alcohol – 2 dips
Ab. alcohol I – 2 dips
Ab. alcohol II – 2 dips
10 Clearing
Finally clear in 2 changes of xylene, 5 min per each to have clear back ground & remove alcohol from tissue sections.
Then you can Mount with mounting media such as DPX
Observe the quality of staining under microscopy.
No responses yet