Re: CNS white matter staining w/ Nissl stain

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From:"L. Gibbs" <>
To:Bret Morrow <>
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To Bret-

I have had the same problem occasionally. It may be a pH problem
with the stain or the tissue. Following ICC and DAB or other
substate (I'm assuming these are not fluorescent probes) I fix
sections with 10% formalin before counterstaining and
dehydrating. Sections should be mounted on slides at this point
and not free-floating sections.

Cresyl will stain "everything" if the pH rises. I use Cresyl
Violet from Roboz (Chrom Gesellschaft) # 1A396. 

5 grams Creyl Violoet
0.2grams sodium acetate
1 liter  DH20

pH to 3.5-3.7 with acetic acid.


Filter stain before use. If staining becomes non-specific, check

You should be able to remove Cresyl with 1% HCL in 70% EtOH.

One more thing, sections should be de-fatted in Xylene prior to
staining with Cresyl. The myelin will be less likely to pick up
the stain if de-fatted. Dehydrate slides slowly to 100% EtOH,
allow to sit in 100% EtOH for 15 minutes, then 2 Changes of
xylene for 15-20 minutes each, hydrate sections to DH20.

Good luck!

Lorraine Gibbs
Physiology & Biophysics
University of Washington

On Fri, 17 Mar 2000, Bret Morrow wrote:

> Greeting,
> I have had the unusual occurrence of having the "white matter" in the
> brains of monkeys and now rats strongly stain with a Nissl stain (Cresyl
> violet or Neutral red).  These brains have already been through a lot:
> the monkey brains were frozen sections while the rat were 4%
> formaldehyde fixed and both types of sections were given at least 1
> immunostain before Nissl staining.  The problem is that occasionally the
> white matter will pick up the Nissl stain and will not let go in spite
> of repeated washes with graded alcohol (70-95%) and the 95% + 0.1%
> Acetic acid.
> The actual Nissl staining of the cortical tissue is OK.  The cortical
> cell layers are where they should be and look good.  The question I have
> is why the heck does this happen occasionally.  I guess more
> importantly: How can I prevent this from happening again?
> ____________________________________________
> Bret A. Morrow, Ph.D.
> Associate research scientist
> Associate clinical professor
> Yale University School of Medicine
> New Haven, CT 06520-8066
> voice 203.785.4515
> fax  203.785.7670
> email

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