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
• Experiment video
• Procedure
• Staining solution
• Maintaining the quality of the staining solution
• Stainability

Staining procedure using Mayer’s hematoxylin solution

1	Deparaffinization	Xylene	3 changes, 10 minuntes each
2	Removal of xylene	100% ethanol	3 changes, 5 minutes each
3	Hydration	95%, 70% ethanol	5 minutes each
4	Washing	Running tap water	2 minutes
5	Rinsing	Distilled water
6	Staining	Hematoxylin solution	4 minutes
7	Blueing	Running tap water	15 minutes
8	Staining	Eosin solution	2 minutes
9	Differentiation	70% ethanol	10 quick dips
10	Dehydration	95% ethanol	Quickly
11	Dehydration	100% ehtanol	3 changes, 5 minutes each
12	Clearing	Xylene	3 changes, 10 minuntes each
13	Coverslipping

⑥ 〜 ⑫ Experiment video

Procedure

Staining solution

Mayer’s hematoxylin solution

hematoxylin	1.0g
distilled water	1000ml
Sodium iodade	0.2g
Either potassium alum or Ammonium alum	50g
Chloral hydrate	50g
Citric acid	1.0g

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-soluble	1.0g
distilled water	100ml

＊ Working solution of eosin

1.0% eosin solution	20ml
80％ ethanol	160ml
acetic acid	10滴

Maintaining the quality of the staining solution

Hematoxylin

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

• Fresh hematoxylin solusion
  A cerebellum specimen stained immediately after preparing the hematoxylin. The color of the Purkinje and granule cell nuclei is blue purple.
• Old hematloxylin solusion
  A cerebellum specimen stained one year after preparing the hematoxylin. The color of the Purkinje and granule cell nuclei is red purple.

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.