Nissl Staining

Pranay Srivastava, Xiqun Chen

Published: 2022-09-14 DOI: 10.17504/protocols.io.bp2l6bz9zgqe/v1

Abstract

This protocol details the staining procedure and mounting procedure of Nissl staining.

Steps

Staining Procedure

Mount sections on positive charged plus slides. Air dry sections .

Incubate the slides directly into 1:1 ethanol/chloroform for 0h 30m 0s.

(All the steps mentioned should be done under the hood)

Incubate in 100% ethanol 0h 2m 0s

Incubate in 95% Ethanol0h 2m 0s

Incubate in 70% Ethanol0h 2m 0s

Incubate in 50% Ethanol0h 2m 0s

Wash in distilled water for 0h 5m 0s

Stain in 0.1% cresyl violet solution for 0h 6m 0s.

Note

Notes: staining in warmed cresyl violet solution (warm up to 37°C-50°C) can improve penetration and enhance even staining. It is particularly beneficial for thicker (30) sections.

Rinse quickly in distilled water.

(check the slides for the best result under the microscope)

10.

Incubate in 50% ethanol (3 dips in the container)

10.1.

Incubate in 95% ethanol (3 dips in the container)

11.

Incubate in 100% ethanol (3 dips in the container)

12.

Incubate in xylene 0h 3m 0s

Mounting Procedure

13.

Pour ~3mL Permount solutioninto a 5mL tube and use a transfer pipette for mounting.

14.

Remove one of the slides from the xylene with tweezer and dry by dabbing it on a paper towel. Then place down flat.

15.

Put 6-7 drops of Permount along the middle of the slide.

16.

Take a cover slip and place the left side down onto the slide.

Note

You want to gently and slowly lower down the right side of the cover slip so that you minimize air bubbles in between the cover slip and the slide. To do so, use a needle to hold the right side, while using the needle's cap to hold the left side down in place.

17.

Once the cover slip is completely on the slide, drain any extra permount on a paper towel and then lay the slides to dry.

18.

If you make a mistake while cover slipping, put the slide with the cover slip back into xylene.

Note

This will remove the permount and the cover slip, allowing you to try again.