In histopathology,the sections (microtomy) as they are prepared are colourless and different components cannot be appreciated. Staining of tissues(histochemical staining) with different coloured dyeshaving affinities for specific components of the tissue, makes identification and study of the tissue morphology possible by histotechnologist. There are somany types of stain such as giemsa stain,pas stain,wright stain,immunofluorescence stain used in immunohistochemistry and Hematoxylin and Eosin (h and e stain) is one of the most frequently used stain in histopathology.Hematoxylin solution or hematoxilin is neither a dye nor it has coloring properties. For nuclear staining with hematoxylin stain, it is necessary to oxidize the hematoxylin to a weak anionic purple dye called hematin
Hematin has no affinity for the nucleic acids of nuclei. Hence the addition of a metallic salt or mordant to hematoxylin gives a positive charge to the dye by virtue of the metal action. Thus the cationic dye (metal complex) will then bind to the anionic nuclear chromatin.
Ammonium or potassium alum ferric salt, chrome alum and phosphotungstic acid are various mordants used. The tissue component which is most frequently demonstrated is the nuclear chromatin using alum mordant in the hematoxylin and eosin staining.
A combination of hematoxylin and mordant is known as hematoxylin lake. The aluminium lake formed with ammonium alum is particularly useful for staining nuclei. Hematoxylin recipes using these mordants are called alum hematoxylin.
How to extract Hematoxylin
- Hematoxylin is extracted from the bark of a tree known as haematoxylum campechianum.
- The hematoxylin which we buy is extracted from this bloodwood tree.
- The bark of freshly logged tree is chipped off, then boil the chips in water which produces an orange red solution which turns yellow, then black on cooling.
- The water is evaporated leaving crude hematoxylin. Further purification is done.
- The solutions of the dye should be oxidized to retain its staining ability longer.
- The dye may also be oxidized by exposing it to the natural light for about 3-4 months.
- As for chemical oxidation,it is achieved by using either sodium iodate or mercuric oxide.
- The chemical oxidation converts the dye almost instantaneously but the product does not have shelf life.
- Sodium iodate is most commonly used oxidizing agent (0.2 gm oxidizes 1.0 gm hematoxylin).
PROPERTIES OF HEMATOXYLIN
- It has no staining property
- The hematin with a mordant such as potassium or ammonium alum forms lake which functions as cationic dye and is able to stains anionic tissue components.
- Also in an aqueous solution, hematin can be an acidic or an alkaline dye depending on pH. 4 of the solution
- Hematin has the affinity for several tissues with an appropriate mordant
Progressive and Regressive staining
It is when the tissue is left in the stain just long enough to reach the proper end point. The slides have to be examined at different interval to find out when the staining is optimum.
With this method, the tissue is overstained then destained (differentiated) until the correct endpoint is reached.An example of a regressive stain is Harris hematoxylin; here,the overstaining is removed using acidified alcohol.The removal of this excess dye is called differentiation.
Because of it’s acid pH,Hematoxylin alum gives a reddish hue to the tissues. To convert this colour to the final blue, an alkaline pH is required and the process is known as “blueing”. Which is done either by tap water or by ammonium hydroxide.
How to prepare Harris’s hematoxylin
The following ingredients are required to prepare Harris’s hematoxylin
|Absolute alcohol||50 ml|
|Ammonium alum||100 gram|
|Distilled water||1000 ml|
|Mercuric oxide||2.5 gram|
|Glacial acetic acid||40 ml|
preparation of harris’s hematoxylin
- Start by dissolving the hematoxylin in absolute alcohol and ammonium alum in hot water.
- Then mix the two solutions and heat in order to boil.
- Remove from flame then add mercuric oxide and cool rapidly.
- Glacial acetic acid if added gives brisk nuclear staining, but life of the solution is reduced. Hence if acetic acid is to be added, it should be added in working solution
How to prepare Mayer’s hematoxylin
The following ingredients are required to prepare Mayer’s hematoxylin
|Distilled water||1000 ml|
|Ammonium alum||50 gram|
|Sodium iodate||0.2 gram|
|Citric acid (reduces pH)||1.0 gram|
|Chloral hydrate (preservative)||50 gram|
Preparation of Mayer’s hematoxylin
- Hematoxylin is dissolved in distilled water using gentle heat.
- Then alum is added and dissolved.
- Then sodium iodate, citric acid and chloral hydrate are added respectively.
Eosin stain or Eosin solution
Eosin stain the cytoplasm to rose colored and is used as a counterstain. The intensity of the eosin is of individual or laboratory choice.
The most widely used eosin is the eosin y stain or eosin yellow where as the “ Y “ stand for yellowish. It is available either in alcohol soluble form or water soluble form.In most laboratories,water soluble form of eosin Y in an alcohol-water solution is used.
Eosin Y water soluble form reagents
|Eosin Y (water soluble)||1.0 gram|
|Distilled water||80 ml|
|95% alcohol||320 ml|
|Glacial acetic acid||0.4 ml|
Preparation of Eosin Y water soluble form
- Start by dissolving eosin in water then add this to 95% alcohol making a one in 5 dilution(one part eosin solution with 4 parts alcohol).
- To the final mixture add a few drops or 0.4 ml of acetic acid .This help to increase the staining intensity of eosin.
- When ready to use, the stain should be cloudy; if the stain is clear, add a few drops of the acetic acid.
- The solution should be standardized by staining with a control slides.
How to stain with Hematoxylin and Eosin stain (h&e staining protocol)
- Start by deparaffinize sections in xylene for 10-20 minutes.
- Filter Hematoxylin.
- Rehydrate sections in 100% alcohol for 1-2 minutes then 95% alcohol for 1-2 minutes
- Rinse in tap water
- Then rinse in distilled water 5
- Now stain with Hematoxylin for a period of 3-5 minutes
- Then wash in tap water
- Differentiate section with 1% HCl in 70% alcohol 1-2 dips and check under microscope. If necessary, return slides to HCl for further differentiation.
- Wash the slides in running tap water for 15 minutes
- Now stain the slides in Eosin for 1-4 minutes
- Dehydration and Differentiation in 95% alcohol 5-6 dips of the slide then 100% alcohol 5-6 dips
- Clear slides in xylene two times
- Mount the slides with Permount or DPX
- At no stage of staining the section should be dry
- H&E is a regressive stain in which a tissue is over-stained and then excess dye is removed to obtain desired intensity of stain
- Remember to filter Hematoxylin each time before staining
- Change most of alcohol and xylene each time before staining