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.

Procedure:

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.

Results:  

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

Positive Controls:

      Brain tissue.