ROUTINE STAINING (H & E)

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ROUTINE STAINING (H & E)
Routine staining is done with haematoxylin and eosin (H&E).



Haematoxylin
This is a natural dye which is obtained from log-wood tree, Haematoxylon campechianum. 
This tree is nowadays commercially grown in Jamaica and Mexico. 
The natural extract from the stem of this tree is haematoxylin which is an inactive product. 
This product is oxidised to an active ingredient which is haematein. 
This process of oxidation is known as ripening which can be done naturally in sunlight, or chemically by addition of oxidant like sodium iodate, KMnO4 or mercuric oxide.
A mordant is added to it (e.g. potash alum) which helps in attaching the stain particles to the tissue.

Procedure for Staining:
Sections are first deparaffinised (removal of wax) by placing the slide in a jar of xylene for 10-15 minutes. 
As haematoxylin is a water-based dye, the sections before staining are rehydrated which is done by passing the sections in a series of descending grades of alcohol and finally bringing the section to water.
􀂄 Place the slide in haematoxylin stain for 8-10 minutes.
􀂄 Rinse in water.
􀂄 Differentiation (i.e. selective removal of excess dye from the section) is done by putting the slide in a
solution of 1% acid alcohol for 10 seconds.
􀂄 Rinse in water.
􀂄 Blueing (i.e. bringing of required blue colour to the section) is done by putting the section in Scott’s tap water (containing sodium bicarbonate and magnesium sulfate) or saturated solution of lithium carbonate for 2-10 minutes.
􀂄 Counterstain with 1% aqueous solution of eosin for 1- 3 minutes.
􀂄 Rinse in tap water.
􀂄 Before mounting, the sections have to be dehydrated which is done by passing the sections in a series of ascending grades of alcohol and finally cleared in
xylene, 2-3 dips in each solution.
􀂄 Mount in DPX (dextrene polystyrene xylene) or Canada balsam.

Results :
Nuclei : Blue
Cytoplasm : Pink
Muscle, collagen, RBCs, keratin, : Pink




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