Nissl Staining Method and Protocol on

Frozen or Vibratome Sections for Brain & Spinal Cord


NovaUltra Special Stain Kits


Description: This method is used for the detection of Nissl body in the cytoplasm of neurons on paraformaldehyde fixed frozen or vibratome tissue sections. The Nissl body will be stained purple-blue. This stain is commonly used for identifying the basic neuronal structure in brain or spinal cord tissue.


Fixation: 4% paraformaldehyde in 0.1M PB or PBS


Section: frozen or vibratome sections at 20-50 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.



  1. Mount frozen or vibratome sections on gelatin coated or positive charged plus slides. Air dry sections or bake slides on slide warmer overnight.

  2. Place slides directly into 1:1 alcohol/chloroform overnight and then rehydrate through 100% and 95 % alcohol to distilled water. DON’T put frozen sections directly to water otherwise they will come off the slides. This is called de-fat step that will reduce background fat staining.

  3. Stain in 0.1% cresyl violet solution for 5-10 minutes. Notes: Staining in warmed cresyl violet solution (warm up in 37-50 ºC oven) can improve penetration and enhancing even staining. It is particularly beneficial for thicker (30 um) sections.

  4. Rinse quickly in distilled water.

  5. Differentiate in 95% ethyl alcohol for 2-30 minutes and check microscopically for best result.  

  6. Dehydrate in 100% alcohol 2x5 min.

  7. Clear in xylene 2x5 min.

  8. Mount with permanent mounting medium.



      Neuron (Nissl body) -------------------------- pink-violet  


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Positive Controls:


      Brain tissue.