⑫ Staining

Ｋlüver-Barrera（KB）staining

Double staining with Nissl staining using cresyl violet and LFB staining of myelin sheaths.

See the Nissl and LFB staining sections for more detailed information on these methods.

• Staining procedure
• Staining results
• Staining pattern

Staining procedure

1	1 Deparaffinization	Xylene	3 changes, 10 minuntes each
2	Removal of xylene	100% ethanol	3 changes, 5 minutes each
3	Hydration	95% ethanol	5 minutes
4	Staining	0.1% luxol fast blue solution	Overnight, 50℃
5	Rinsing	95% ethanol	1 minute
6	Washing	Running tap water
7	Differentiation	0.05% lithium carbonate solution	5 seconds to 20 seconds
8	Differentiation	70% ethanol	3 changes. 1 minute each in the first and second dish. Leave sections in the third dish for approximately 1 to 10 minutes.
9	Microscopic check		Repeat step ⑦&⑧, until gray matter become white.
10	Washing	Running tap water
11	Rinsing	Distilled water
12	Staining	0.1% cresyl violet solution	10 minutes, 37℃
13	Differentiation	95% ethanol + a few drops of 10% acetic acid solution	Approximately 5 to 10 minutes. Care should be taken not to overdifferentiate since decolorization will proceed in the following step
14	Differentiation	95%, 100% ethanol	Approximately 1 minute each. Differentiate until only nuclei and nissl bodies are blue purple.
15	Microscopic check		Step ⑦ & ⑧ may be repeated if more differentiation is needed.
16	Dehydration	100% ehtanol	2 changes, 5 minutes each
17	Clearing	Xylene	3 changes, 10 minuntes each
18	Coverslipping

Staining results

The color tones of neurons and myelin sheaths are more distinct with KB staining compared with LFB or Nissl staining alone. The change in color tone is particularly notable for the myelin sheaths. With LFB staining, the myelin sheath is stained emerald green, whereas it is stained navy blue with KB staining, which creates a sharper contrast.

• 
  Nissl staining
• 
  LFB staining
• 
  KB staining

Staining pattern

In a Loupe image, the gray matter is stained a pale, cloudy blue color and the white matter is stained bright blue, which improves the detection of white matter lesions.

Nerve cell body

Normal histology	Pathology
Nissl substance- blue purple	Achromasia - pale blue color with loss of nissle substance
Lipofuscin - yellowish brown
Neuromelanin - Dark brown

Nerve process

Normal histology	Pathology
Unstained	Unstained

Astrocyte

Normal histology	Pathology
Unstained	Rosenthal fiber-blue

Oligodendrocyte

Normal histology	Pathology
Unstained	Unstained

Myelin sheath

Normal histology	Pathology
The sheath wrapped around the axon-blue	Demyelinated areas are unstained.

Macrophage

Normal histology	Pathology
Unstained	Myelin fragments recently engulfed by macrophages - blue
	Fat granule cell - colorless
	Iron granule cell - brown