⑫ Staining

Nissl staining

Nuclei and nissl bodies (rough endoplasmic reticulum) are stained blue purple with cresyl violet.

Nissl staining is used for Klüver-Barrera (KB) staining.

Staining procedure

1DeparaffinizationXylene3 changes, 10 minuntes each
2Removal of xylene100% ethanol3 changes, 5 minutes each
3Hydration95%, 70% ethanol5 minutes each
4WashingRunning tap water2 minutes
5RinsingDistilled water 
6Staining0.1% cresyl violet solution10 minutes, 37℃
7Differentiation95% 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.
8Differentiation95%, 100% ethanolApproximately 1 minute each. Differentiate until only nuclei and nissl bodies are blue purple.
9Microscopic check Step ⑦ & ⑧ may be repeated if more differentiation is needed.
10Dehydration 100% ehtanol2 changes, 5 minutes each
11ClearingXylene3 changes, 10 minuntes each

0.1% cresyl violet aqueous solution (Filter before use)

cresyl violet1g
distilled water1000ml

pH of cresyl violet

The staining method differs depending on the pH of the staining solution. At a pH close to 3.0, only the Nissl bodies, nucleoli, and nuclear membranes are stained pale blue. Glial cell nuclei are only very slightly stained. As the pH increases, the color becomes darker overall, and the cytoplasm of nerve cells, nerve fibers, and glial cells are co-stained. Co-staining intensifies close to a pH of 4.0. Therefore, a longer differentiation time is required.

Types of cresyl violet

Three types of cresyl violet are currently used in the Laboratory of Neuropathology.

product namemanufactureravailability
Cresyl Violet AcetateEM SCIENCEout of stock
Cresyl Violet acetateSIGMAAvailable commercially
Cresyl violet (Acetate)MERCKAvailable commercially

Differentiation time differs depending on the cresyl violet product

The pH of the staining solution differs depending on the product. Therefore, the staining methods also differ. It is important to be aware that the time required for differentiation differs between products. However, whichever product is used, the same stained chromatic image can ultimately be obtained through differentiation and observation of the specimen under a microscope.

product namemanufacturerpH of 0.1% cresyl violet solutionDifferentiation time in 95% ethanol
Cresyl Violet AcetateEM SCIENCE3.5for 10 to 30 seconds
Cresyl Violet acetateSIGMA4.710min approximately
Cresyl violet (Acetate)MERCK4.810min approximately

※ The pH can be adjusted from 3.5 to approximately 3.8 by adding a suitable amount of 10% acetic acid to the Sigma and Merck staining solutions, which will shorten the time needed for differentiation with 95% ethanol. However, it is not recommended to reuse this solution.

Important considerations regarding differentiation

Perform differentiation until the background turns white. Having the nucleoli and nuclear membranes of the nerve cell nuclei clear and chromatin faintly stained is ideal.

Cresyl Violet acetate (Sigma C5042) pH4.7 was used in the both sections.

Left photograph: 95% ethanol differentiation for only 10 seconds. With insufficient differentiation, the entire nerve cell is stained blue, making it impossible to identify the nucleus and Nissl bodies.

Right photograph: 95% ethanol differentiation for 10 minutes. Sufficient differentiation allows the nuclei and Nissl bodies of neurons to be clearly differentiated.