Description

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

Purpose: Nissl staining uses a cresyl violet solution to stain RNA , 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 H2 O mixture

Xylene

Resin medium

1) Prepare 0.1% cresyl violet solution by mixing 0.1 g cresyl fast violet mixed in 100 mL deionized H2 O. 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 H2 O 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 for staining .

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 for staining . Nissl bodies (neurons) will be stained red-violet.