Nissl Staining for Neurons

From the lab of Lucien T. "Tres" Thompson, Ph.D. The University of Texas at Dallas

NisslStainingfor Neurons

This is a general protocol adapted from a protocol on ihcworld.com.

Purpose:Nisslstaininguses a cresyl violet solution to stainRNA, which is most abundant in the rough endoplasmic reticulum of nuclei. This method is used to detect the nuclei of neurons in fixed, embedded, and frozen tissue.

Materials

30-50 μm fixed frozen/vibratome sections, mounted on slides

Beakers to mix and hold solutions

Incubation chamber

Solutions

Cresyl fast violet

Glacial acetic acid

100% ethyl alcohol

Chloroform

95% ethyl alcohol / 5% deionized H₂O mixture

Xylene

Resin medium

1)Prepare 0.1% cresyl violet solution by mixing 0.1 g cresyl fast violet mixed in 100 mL deionized H₂O.Add 10 drops of glacial acetic acid just before use and filter.

2)De-fat the tissues by soaking in a 1:1 alcohol/chloroform mixture overnight.

3)Rehydrate the slices in 100% alcohol followed by a 95% alcohol / 5% deionized H₂O mixture.Note:Putting the slides in pure water would cause the frozen tissue to come off the slides.

4)Stain in cressyl violet for 3-5 minutes.

5)Rinse in distilled water.

6)Soak in 95% ethyl alcohol for 5-30 minutes.Check microscopically forstaining.

7)Dehydrate in 100% alcohol for five minutes.Replace alcohol and repeat.

8)Clear in xylene for five minutes.Replace xylene and repeat.

9)Mount with resin medium.

Check forstaining.Nissl bodies (neurons) will be stained red-violet.