⑫ Staining

Hematoxylin and Eosin(HE)staining

Hematoxylin stains the cell nucleus blue purple, whereas eosin stains other structures in various shades of red.

Staining procedure using Mayer’s hematoxylin solution

1DeparaffinizationXylene3 changes, 10 minuntes each
2Removal of xylene100% ethanol3 changes, 5 minutes each
3Hydration95%, 70% ethanol5 minutes each
4WashingRunning tap water2 minutes
5RinsingDistilled water 
6 StainingHematoxylin solution4 minutes
7BlueingRunning tap water15 minutes
8StainingEosin solution2 minutes
9Differentiation70% ethanol10 quick dips
10Dehydration95% ethanolQuickly
11Dehydration 100% ehtanol3 changes, 5 minutes each
12ClearingXylene3 changes, 10 minuntes each

⑥ 〜 ⑫ Experiment video


Staining solution

Mayer’s hematoxylin solution

distilled water1000ml
Sodium iodade0.2g
Either potassium alum or Ammonium alum50g
Chloral hydrate50g
Citric acid1.0g

There are many types of hematoxylin solutions. The typical hematoxylin solutions are those of Mayer and Carracci.

Mayer’s staining solution contains acid, whereas Carracci’s hematoxylin is relatively neutral because it does not contain acid. Therefore, the staining methods differ between the Mayer and Carracci stains.

With Mayer’s hematoxylin, the nucleus is stained red purple at first and then turns blue purple under running water. Conversely, with Carracci’s hematoxylin, the entire tissue is stained blue purple and then differentiated with hydrochloric acid-alcohol solution, resulting in only the nucleus retaining the blue-purple stain.

Eosin solution

*1.0% eosin solution(stock solution)
Eosin Y, water-soluble1.0g
distilled water100ml
* Working solution of eosin
1.0% eosin solution20ml
80% ethanol160ml
acetic acid10滴

Maintaining the quality of the staining solution


After preparing the staining solution, the staining results change as the stain is used multiple times. The following three explanations may be related to this change:

  1. The natural oxidation of hematoxylin.
  2. The staining solution becomes contaminated with water which is carried with a section.
  3. The concentration of the staining solution decreases as the pigment is consumed.

It is essential to regularly change the staining solution to maintain consistent staining results. Refrigerating the staining solution makes the solution last longer; however, the solution must be at room temperature when used.

Staining deterioration due to the natural oxidation of hematoxylin

Eosin solution

Unlike hematoxylin, eosin still stains well even when the solution is old.

When a stronger stain is needed, adding a small amount of acetic acid is effective.

Staining pattern

Please note that, although almost all normal and abnormal structures can be observed with HE staining, some structures that are important for differential diagnosis cannot be visualized using this staining method (see table below).
Therefore, these structures require special staining or immunostaining.
Basophilic appearance differs slightly depending on the hematoxylin solution used (e.g., Carracci or Mayer).

Nerve cell body

Normal histology Pathology Structures invisible in H&E preparations
  • Cytoplasm (the non-nucleotides containing organelles)
  • Almost every kinds of inclusion bodies - various shades of pink and various shapes
  • Ubiquitinated inclusions in ALS-D
  • Skein

detected by TDP-43 and ubiquitin immunostaining
  • Polyglutamine immunoreative intranuclear inclusions

detected by polyglutamine immunostaining)
  • Nissl substance
  • Nuclear membrane
  • Nucleous and chromatin(the nucleotides containing structures)
  • Basophilic inclusions in a variety of neurodegenerative disorders like FTLD-FUS
  • Some types of NFT
  • Polyglucosan body (lafora body)
  • Lipofuscin - yellowish brown
  • Neuromelanin - Dark brown

Nerve process

Normal histologyPathology
  • Structures invisible in H&E preparations
  • Difficult to discern (Nerve processes proximate to the cell body can be discerned.)
  • Spheroids - various shades of pink and various shapes
  • Abnormalities in Purkinje dendrites (cactus, asteroid body)
  • Glomerular structure in hypertrophic olivary degeneration
  • Lewy neurite
  • Grumose degeneration
  • Dystrophic neurite in senile plaque
  • Neuropil threads
  • Argyrophilic grains

detected by gallyas-braak staining and tau immunostaining


Normal histologyPathologyStructures invisible in H&E preparations
  • Cytoplasm & cellular processes (scanty)
  • Gemistocytic astrocyte
  • Fibrillary astrocyte
  • Gliosis
  • Rosenthal fiber
  • Intranuclear viral inclusions
  • Alzheimer's type 1 astrocyte (type2 is basophilic)
  • Dysplastic astrocytes
  • Foamy spheroid body
  • Tuft-shaped astrocytes
  • Astrocytic plaque
  • Thorn-shaped astrocytes

detected by tau - immunostaining and gallyas - braak staining
  • Nuclear membrane
  • nucleolus
  • chromatin
  • Corpora amylacea - grayish blue


Normal histologyPathologyStructures invisible in H&E preparations
  • Cytoplasm (scanty)
  • Glial cytoplasmic inclusions in MSA -pale pink
  • Intranuclear viral inclusions by measles viruses (SSPE)
  • Glial coiled body(detected by gallyas-braak staining and tau immunostaining)
  • Nucleus (lymphocyte-like)
  • Intranuclear viral inclusions by JC viruses (PML)

Myelin sheath

Normal histologyPathology
  • Difficult to discern
  • Difficult to discern


Normal histologyPathology
  • Nucleus
  • Rod-shaped microglia


Normal histologyPathology
  • Nucleus
  • Myelin fragments recently engulfed by macrophages
  • Cytoplasm - colorless
  • Fat granule cell - colorless
  • Iron granule cell - brown
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