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.

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