Nissl Staining Protocol for Cryosections       

Nissl staining protocol for cryosections post we have briefly explained about Nissl staining method’s principle, requirements, nissl staining protocol procedure, result with Label for nissl staining protocol.

Nissl Staining Protocol for Cryosections

Cresyl Violet Acetate solution is used to stain Nissl material in the cytoplasm of neurons in paraformaldehyde or formalin-fixed tissue. The neuropil will be stained with a granular purple-blue stain. This nissl staining protocol is commonly used to determine neuronal anatomy in brain and spinal cord tissue.

Principle

By staining RNA blue with a basic aniline dye, the Cresyl Violet method shows important structural characteristics of neurons. The Nissl material (rough endoplasmic reticulum) appears dark blue due to ribosomal RNA staining, giving the cytoplasm a mottled appearance. Individual extranuclear RNA granules are known as Nissl granules (ribosomes). The staining colour of DNA in the nucleus is identical.

Requirements

Sections

1. Fixation: 4% paraformaldehyde in 0.1M PB or PBS

2. Section: Floating sections at 20-50 um.

Reagents

1. 95% Ethanol

2. 70% Ethanol

3. Cresyl Violet Acetate 0.2%

Cresyl Violet

1. Cresyl Violet (or Cresyl violet acetate) Add 0.1 g to 100 mL of distilled water Just before using, add 10 drops (or 0.3 ml) of glacial acetic acid and filter.

Nissl Staining Protocol

1. To eliminate any leftover salts, run slides under cold water for a few seconds. Immerse slides in 100 percent ethanol for 2×3 min changes in nissl staining protocol.

2. Remove the fat from the tissue: 15 minutes in 100% xylene (2-3 changes as directed), then 10 minutes in 100% ethanol. Rehydrate for 3 minutes each with alcohol (100 percent x2). Use tap water to clean.

3. Nissl staining protocol staining process takes 4-15 minutes. Give it a brief rinse with tap water to get rid of any remaining stains.

4. Ethanol washes with 70% ethanol (the stain will be removed by this method). Absolute ethanol changes for 2×3 minutes dry the body.

5. Allow at least 48 hours or overnight to dry in the fume hood for optimal results. After clearing in xylene, mount in DPX in nissl staining protocol.

Results

Nissl staining

Nissl Staining Protocol: Neuron (Nissl body): pink-violet 

Further Readings

Reference