The most common histological staining method is discussed in detail with troubleshooting ideas

1. Haematoxylin & Eosin

2. • Most widely used histological stain.
• Simplicity and wide range of applications.
• Show most histological structures.
• Particularly suited to demonstrate nucleus.
• Provides information to indicate which other staining
methods are to be used.

3. Haematoxylin
• Extracted from the heart-wood -Haematoxylin
campechianum.
• Pure Haematoxylin: Colourless; oxidized in air
‘Haematein’.
• Haematein: poor dye.
• Metallic mordant: forms a powerful stain.

4. Haematoxylin
Haematein`

5. Oxidation of Haematoxylin
Natural Oxidation (Ripening)
– Exposure to sunlight and air for about 6-8 weeks or air bubbled
through the solution for 3-4 weeks.
– Advantage: Long shelf-life; lesser fading of slides.
– Disadvantage: Slow process.
Chemical Oxidation
– Artificial ripening: addition of oxidizing agents. Eg. HgO, NaI, KI
etc.
– NaI preferred - does not require boiling, and increased shelf-life.
– Advantage: Used as soon as it is made
– Disadvantage: Has shorter shelf-life.

6. Types of Haematoxylin
• Alum Haematoxylin
• Iron Haematoxylin
• Tungsten Haematoxylin
• Molybdenum Haematoxylin
• Lead Haematoxylin
• Haematoxylin without Mordant

7. Alum Haematoxylins

8. • Comprises most of the hematoxylins used
routinely in H&E stains.
• Mordant is usually in the form of potash alum or
ammonium alum.
• Blueing:
– When stained with an Al haematoxylin, nuclei are a
dark red colour
– To change it to blue and to stabilize the dye, sections
treated with a weak alkali
– Tap water, if alkaline, may be used

9. • Mayer’s H (Mayer 1903)
• Harris’s H (Harris 1900)
• Ehrlich’s H (Ehrlich 1886)
• Delafield’s H (Delafield 1885)
• Carazzi’s H (Carrazzi 1911)
• Gill’s H (Gill et al. 1974)
• Cole’s H (Cole 1943)

10. Mayer’s Haematoxylin
Haematoxylin 1 g.
Distilled water 1000 ml.
Ammonium alum 50 g.
Sodium iodate 0.2 g.
Citric acid 1 g.
Chloral hydrate 50 g.
• Dissolve H in distilled water using gentle heat. Add the alum,
shaking to dissolve, followed by the sodium iodate. Continue
heating whilst adding the citric acid and chloral hydrate. The stain
is ready for use when cooled.
– Ammo.alum: Mordant
– NaI: Oxidant
– Chloral hydrate: Preservative
– Citric Acid: Acidifies sol and sharpens staining

11. • As a regressive stain like all alum H.
• Very useful as a progressive stain, particularly as a
nuclear counter-stain for cytoplasmic special stains.
• Situations where acid-alcohol differentiation might
destroy or de-color the special stain.
• Eg. PAS, Mucicarmine and many enzyme histochemical
procedures.

12. PAS Staining for Goblet Cells

13. Harris’s Haematoxylin
Haematoxylin 2.5 g.
Absolute alcohol 50 ml
Ammonium alum 50 g
Distilled water 500 ml
Mercuric oxide 1.5 g.
Glacial acetic acid 20 ml
• Dissolve the haematoxylin in the alcohol and the alum in hot water.
Mix the two solutions together and heat to boiling. Add the mercuric
oxide and cool rapidly. Glacial acetic acid, added after cooling.
– Mercuric oxide: Oxidant (Toxic. So replaced by NaI or KI)
– Glacial Acetic acid: Acidify sol and sharpen nuclear staining

14. • Popular method for routine use.
• Does not need time to ripen.
• Results are consistent and rapid.
• Regressive stain in routine histology. Usually
differentiated in Acid-alcohol (1% HCl in 70% Alcohol).
• If used progressively, use milder differentiating agent like
acetic acid/alcohol for better control.

15. Common Errors with Harris’s H
• Dark grey or black granules - filter stain once a week.
• Dull red or brown staining of nuclei - over-ripe H or
insufficient blueing.
• Nuclei appear as blue rings with colourless centres - short
staining time.
• Uniformly weak staining of the nuclear membrane and
chromatin - over-differentiation.
• Nuclei are blue and congested without proper chromatin
detail - insufficient differentiation.

16. Ehrlich’s Haematoxylin
Haematoxylin 6 g.
Absolute alcohol 300 ml.
Distilled water 300 ml.
Glycerol 300 ml.
Glacial acetic acid 30 ml.
Potassium alum (to saturation) 10-14 gm
• Haematoxylin should be fully dissolved in the alcohol before the other
ingredients are added. Potassium alum is added until saturation. The
solution is allowed to ripen in clear glass bottles over 6-8 weeks. Artificial
ripening by the addition of 0.9 g NaI for immediate use.
– No Chemical oxidant: natural ripening
– Glycerol: Stabilizes against over-oxidation and reduces
evaporation.
– Retardant effect on dye action. So more staining time needed.

17. • Strong haematoxylin: stains nuclei intensely.
• Mucin in salivary glands and goblet cells.
• Cells and the ground substance of cartilage.
• High H content:
– Staining of decalcified bone sections (preferred)
– Stains sections from tissue which has been stored too
long in non-buffered formalin fixatives.

18. • Advantages:
 Fine nuclear chromatin is shown more precisely.
 Less liable to fading.
 Staining of difficult tissue sections
• Disadvantages:
 The long period needed for ripening.
 The length of time required for staining (45 min).
 Unsuitable for frozen sections because of its high alcohol
content.
 High concentration of haematoxylin makes it expensive.

19. • Delafield’s Haematoxylin
– Similar to Ehrlich’s: ripens naturally and thus will last
indefinitely.
• Carazzi’s Haematoxylin
– Similar to Mayer’s.
– KI is the oxidant.
– Used as a progressive nuclear counterstain in special stains. Eg.
PAS, Congo red
– Pale precise nuclear stain that does not stain any of the
cytoplasmic components.
– Urgent staining of frozen sections: Double strength Carrazzi’s
haematoxylin (1min).

20. • Gill’s Haematoxylin
– Fast action; stable for atleast 12 months.
– Preparation does not involve boiling.
– Major disadvantage is excessive background staining:
it stains the gelatin adhesive and even the glass itself.
– Aluminium sulfate (mordant): responsible for this
effect.
• Cole’s haematoxylin
– Good keeping qualities, but will require filtering before
use.

21. Staining with Haematoxylin
• Principle: Acid-base interaction.
– Basic nucleus: stains with acidic haematoxylin
– Acidic cytoplasm: stains with basic eosin
• All nuclear staining schedules: used as a regressive stain.
• 2 exceptions:
– For urgent methods.
– As a counterstain following a stain that would be
removed if the haematoxylin is differentiated.

22. Staining Times with Alum
Hematoxylins
• Type of Haematoxylin: eg. Ehrlich’s H - 45min, Harris’s H -10-15min.
• Age of Stain: Staining time will need to be increased for older sol.
• Degree or Usage: heavy usage - quicker loss of staining power -increased
staining time.
• Whether used progressively or regressively: eg. Mayer’s H used
progressively- 5-10min; used regressively- 10-20min.
• Pre-treatment of tissues: long time in fixative, acid decalcified tissues etc
need longer staining time. Frozen sections need less staining time.
• Post-treatment of sections: eg. Subsequent acid stains such as Van Gieson
remove hematoxylin. Increase staining time with alum H or use other H.
• Personal preference.

23. Eosin
• A xanthene dye: tetrabromofluorescein.
• Water soluble Eosin Y is the most widely used.
• Used as a 0.5% or 1% solution in water. Thymol prevents fungal
growth. CH3COOH or CaCl2 improves stain performance...
• Most suitable stain to combine with an Alum H to demonstrate the
general histological architecture of a tissue.
• Ability to distinguish between the cytoplasm of different types of
cells, and between the different types of connective tissue fibers and
matrices.
• Staining: differing shades of pink and red.

24. • Differentiation of Eosin: tap water wash +
dehydration through alcohols.
• Intensity of eosin staining and the degree of
differentiation: individual preference.
• Used as a counter stain for haematoxylin.
• Specific stain for RBC’s and eosinophils.

25. Standard H&E Staining
• Dewax sections and hydrate.
• Stain with Alum H of choice.
• Wash in running water.
• Differentiate in acid alcohol (1% HCl
in 70% Alcohol). Wash.
• Blueing in tap water or ammonia
water. Wash.
• Stain with 1% Aq. Eosin: 3-10min
• Wash in running water: 5-10min
• Dehydrate, clear & mount.
Results
 Nuclei, RNA rich cytoplasm,
calcium - Blue
 Cytoplasm - Varying shades of
pink
 Muscle, fibrin, Keratin,
Collagen - Deep Pink/Bright
red
 RBC’s - Orange/ red.

26. H&E Oesophagus

27. The Celestine blue-Alum H
Procedure
• Disadvantage of Alum H’s: sensitivity to any subsequent applied
acidic staining solutions.
• Eg. Van Gieson and other trichome stains. The application of the
Picric acid-Acid fuschin mixture in Van Gieson stain removes most of
the hematoxylin.
• The solution: use stronger H’s like Iron H.
• Or use the Celestine Blue/Alum H procedure.
• More recent, suitable and currently more popular method.

28. • Celestine blue sol: Celestine blue B + Ferric Ammo
sulphate.
• Celestine blue stains nucleus. Resistant to the effects of
acid.
• Ferric salt strengthens the bond between the nucleus and
alum H.
• A strong nuclear stain which is reasonably resistant to
acid used subsequently.
• Treat sections with Celestine blue sol for 5min before
proceeding with Alum H staining.

29. Celestine blue/Alum H staining

30. Iron Haematoxylins

31. • Use iron salts as both the oxidizing agent and the
mordant.
• Ferric Chloride and Ferric Ammonium Sulfate are
commonly used.
Weigert’s H (Weigert 1904)
Heidenhain’s H (Heidenhain 1896)
Loyez H for Myelin
Verhoeff’s H for Elastic fibers

32. • Over-oxidation: problem with iron H’s.
• Prepare separate Iron salt and haematoxylin solutions.
• Mix them immediately before use eg. Weigert’s H, or use them
consecutively eg. Heidenhain’s.
• Iron salts solutions also used as subsequent differentiating fluid
after H staining.
• Iron H’s: strong nuclear stain; capable of demonstrating a much
wider range of tissue structures than alum H.
• Techniques are time consuming and usually incorporate a
differentiation stage which needs microscopic control for accuracy.

33. Weigert’s Haematoxylin
Solution A
Haematoxylin 1g.
Absolute alcohol 100 ml.
– Use gentle heat to dissolve
Solution B
30% aqueous ferric chloride 4 ml.
Conc. Hcl 1 ml.
Distilled water 100 ml.
• Mix equal quantities of A and B immediately before use. The mixture should
be a violet black color and must be discarded it it is brown.

34. • Sometimes used with eosin especially
for sections of brain.
• Chief use: methods where it is used as
a nuclear counterstain. Eg. Van Gieson’s and
other Trichome stains.
.

35. Van-Gieson Stain

36. Heidenhain’s Haematoxylin
• The most intense of haematoxylins.
• Used as a regressive stain.
• The iron alum solution is used as both mordant and
differentiator.
• Counterstains are unnecessary.
• Resistant to fading provided that iron alum has been
thoroughly removed by washing.

37. • Produces a dark grey or black colour.
• What structures are stained will depend on the degree of
differentiation.
– Mitochondria are decolorized quickly.
– Muscle cross-striations and nuclei relatively resistant to differentiation.
• Can be used for staining of sections that reject other haematoxylin
mixtures. Eg. tissue fixed in osmium tetroxide, tissue in decalcifying
fluid, or was stored for a very long time in fixative or in alcohol.
• It may be used with orange G for demonstration of mitoses and other
fine nuclear detail.

38. Muscle Striations (Heidenhain’s H)

39. Limitations:
• Tissue section needs to be treated with the mordant solution
first followed by the haematoxylin. The length of time in each
is usually very long (1hour-24hours).
• The degree of differentiation needs to be microscopically
controlled until desired structure is viewed satisfactorily.
• If the differentiation proceeds too far then staining has to be
started again.
• Residual iron solution left on slide: quick fading of slides.

40. Other Iron Haematoxylins
• Loyez Haematoxylin
– Used to demonstrate myelin.
– Iron alum is the mordant used.
• Verhoeff’s Haematoxylin
– Used to demonstrate elastic fibers.
– Ferric chloride with Lugol’s Iodine is the
mordant.

41. Tungsten Haematoxylin
– Most widely used: Mallory’s phosphotungstic acid
haematoxylin (PTAH)
– Connective tissue stain useful for demonstration of muscle
striations and fibrin.
Molybdenum Haematoxylin
– Used for the demonstration of collagen and coarse reticulin.
– Better techniques for these exist.
Lead Haematoxylins
– Used in the demonstration of granules in the endocrine cells of the
alimentary tract and other regions.

42. HAEMATOXYLIN without MORDANT
• Mallory and Parker:
– Used freshly prepared haematoxylin solution for demonstrating
minerals like Copper, Iron and Lead in tissue sections.
– Based on the ability of unripened haematoxylin to form blue/black
lakes with these metals.
• Weigert-Pal Technique:
– Used for myelin.
– Does not use a mordant with the haematoxylin.
– But the tissue block is mordanted in a chromate solution before
embedding and sectioning.
– So not a true un-mordanted haematoxylin in strict sense.

43. Conclusion
• Haematoxylin is probably the single most important dye
employed in histological staining.
• Acts as a morphological reference in many specialized
histochemical methods.
• Not limited to nuclear staining.
• Understanding the various types of haematoxylin and their
specific uses will help in more judicious and informative
use of these in diagnostic pathology.