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
• Important considerations regarding differentiation

Staining procedure

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	0.1% cresyl violet solution	10 minutes, 37℃
7	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.
8	Differentiation	95%, 100% ethanol	Approximately 1 minute each. Differentiate until only nuclei and nissl bodies are blue purple.
9	Microscopic check		Step ⑦ & ⑧ may be repeated if more differentiation is needed.
10	Dehydration	100% ehtanol	2 changes, 5 minutes each
11	Clearing	Xylene	3 changes, 10 minuntes each
12	Coverslipping

0.1% cresyl violet aqueous solution (Filter before use)

cresyl violet	1g
distilled water	1000ml

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.

• 
  pH3.0
• 
  pH4.7

Types of cresyl violet

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

product name	manufacturer	availability
Cresyl Violet Acetate	EM SCIENCE	out of stock
Cresyl Violet acetate	SIGMA	Available commercially
Cresyl violet (Acetate)	MERCK	Available 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 name	manufacturer	pH of 0.1% cresyl violet solution	Differentiation time in 95% ethanol
Cresyl Violet Acetate	EM SCIENCE	3.5	for 10 to 30 seconds
Cresyl Violet acetate	SIGMA	4.7	10min approximately
Cresyl violet (Acetate)	MERCK	4.8	10min 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.