⑫ 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

11 DeparaffinizationXylene3 changes, 10 minuntes each
2Removal of xylene100% ethanol3 changes, 5 minutes each
3Hydration95% ethanol5 minutes
4Staining0.1% luxol fast blue solution Overnight, 50℃
5Rinsing95% ethanol1 minute
6WashingRunning tap water 
7Differentiation0.05% lithium carbonate solution5 seconds to 20 seconds
8Differentiation70% ethanol3 changes. 1 minute each in the first and second dish. Leave sections in the third dish for approximately 1 to 10 minutes.
9Microscopic check Repeat step ⑦&⑧, until gray matter become white.
10WashingRunning tap water 
11RinsingDistilled water 
12Staining0.1% cresyl violet solution10 minutes, 37℃
13Differentiation95% ethanol + a few drops of 10% acetic acid solutionApproximately 5 to 10 minutes. Care should be taken not to overdifferentiate since decolorization will proceed in the following step
14Differentiation95%, 100% ethanolApproximately 1 minute each. Differentiate until only nuclei and nissl bodies are blue purple.
15Microscopic check Step ⑦ & ⑧ may be repeated if more differentiation is needed.
16Dehydration100% ehtanol2 changes, 5 minutes each
17ClearingXylene3 changes, 10 minuntes each

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.

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 purpleAchromasia - pale blue color with loss of nissle substance
Lipofuscin - yellowish brown
Neuromelanin - Dark brown

Nerve process

Normal histology Pathology


Normal histology Pathology
UnstainedRosenthal fiber-blue


Normal histology Pathology

Myelin sheath

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


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