Sep 14, 2022

Public workspaceNissl Staining

DOI
dx.doi.org/10.17504/protocols.io.bp2l6bz9zgqe/v1
  • 1MassGeneral Institute for Neurodegenerative Disease, Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, USA Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, Towson, MD, USA
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DOI: dx.doi.org/10.17504/protocols.io.bp2l6bz9zgqe/v1
Protocol Citation: Pranay Srivastava, Xiqun Chen 2022. Nissl Staining. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6bz9zgqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 15, 2021
Last Modified: May 31, 2024
Protocol Integer ID: 50824
Keywords: Nissl Staining, Staining Procedure, Mounting Procedure, ASAPCRN
Abstract
This protocol details the staining procedure and mounting procedure of Nissl staining.
Materials
Solutions and Reagents:

0.1% Cresyl violet solution:
AB
Cresyl echt violet (or cresyl violet acetate)0.1g
Distilled water100 ml
Add 10 drops (0.3 ml) of glacial acetic acid just before use and filter.

Staining Procedure
Staining Procedure
1h 19m
1h 19m
Mount sections on positive charged plus slides. Air dry sections DurationOvernight .

Overnight
Incubate the slides directly into 1:1 ethanol/chloroform for Duration00:30:00 .
(All the steps mentioned should be done under the hood)
30m
Incubate in 100% ethanol Duration00:02:00

2m
Incubate in 95% EthanolDuration00:02:00

2m
Incubate in 70% EthanolDuration00:02:00
2m
Incubate in 50% EthanolDuration00:02:00
2m
Wash in distilled water for Duration00:05:00

5m
Stain in 0.1% cresyl violet solution for Duration00:06:00 .
Note
Notes: staining in warmed cresyl violet solution (warm up to Temperature37 °C -Temperature50 °C ) can improve penetration and enhance even staining. It is particularly beneficial for thicker (Amount30 µL ) sections.


6m
Rinse quickly in distilled water.
(check the slides for the best result under the microscope)
Wash
Incubate in 50% ethanol (3 dips in the container)
Incubate in 95% ethanol (3 dips in the container)
Incubate in 100% ethanol (3 dips in the container)
Incubate in xylene Duration00:03:00

3m
Mounting Procedure
Mounting Procedure
Pour ~Amount3 mL Permount solutioninto a Amount5 mL tube and use a transfer pipette for mounting.

Pipetting
Remove one of the slides from the xylene with tweezer and dry by dabbing it on a paper towel. Then place down flat.
Put 6-7 drops of Permount along the middle of the slide.
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.


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