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Nissl Staining Method and Protocol on
Paraffin Sections for Brain & Spinal Cord
Description: This method is used for the detection of Nissl body in the cytoplasm of neurons on paraformaldehyde or formalin-fixed, paraffin embedded tissue sections. The Nissl body will be stained purple-blue. This stain is commonly used for identifying the basic neuronal structure in brain and spinal cord tissue.
Fixation: 4% paraformaldehyde in 0.1M PB or 10% formalin.
Section: paraffin sections at 5~30 um
Solutions and Reagents:
0.1% Cresyl violet solution:
Cresyl echt
violet (or cresyl violet acetate) --- 0.1 g
Distilled water
------------------------------------ 100 ml
Add 10 drops (or 0.3 ml) of glacial acetic acid just before use and filter.
Procedure:
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Deparaffinize sections in xylene 2 or 3 changes at 10 minutes each. Hydrate in 100% alcohol 2x5 minutes, 95% alcohol 3 minutes, 70% 3 minutes. Rinse in tap water and then in distilled water. Notes: For 5-10 um sections, 2 changes of xylene, 10 minutes each, will be sufficient. However, for 20-50 um sections, 3 changes of xylene at 10 minutes each, is recommended. Insufficient deparaffinization will cause uneven staining.
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Stain in 0.1% cresyl violet solution for 3-10 minutes.
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Rinse quickly in distilled water.
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Differentiate in 95% ethyl alcohol for 2-30 minutes and check microscopically for best result.
-
Dehydrate in 100% alcohol 2x5 min.
-
Clear in xylene 2x5 min.
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Mount with permanent mounting medium.
Results:
Neuron (Nissl
body) -------------------------- pink-violet
Brain tissue.