<!doctype html public "-//W3C//DTD W3 HTML//EN">
<html><head><style type="text/css"><!--
blockquote, dl, ul, ol, li { margin-top: 0 ; margin-bottom: 0 }
--></style><title>FW: Cresyl Violet Staining</title></head><body>
<div>Dear Lorraine and Sarah,</div>
<div><br></div>
<div>First, where is Sarah and what is her email address?
Sounds as if she could benefit from some local hands-on advice, and
from the HistoNet email list. (Subscribing to the HistoNet
Daily Digest results in one message each morning rather than a slew
of 20-50 messages throughout the day). </div>
<div><br></div>
<div>Following is a long post on this specialty topic--apologies to
those not interested...</div>
<div><x-tab> </x-tab></div>
<div>In staining 30-50micron thick frozen sections of mammalian brain
with cresyl violet acetate or other stains for Nissl substance, it is
necessary to "defat" the sections first, unless there is
some over-riding biochemical or experimental reason not to do
so. The staining protocol you give sounds like one for very
thin sections, or for preserving a labile histochemical reaction
product while visualizing neurons in thick frozen sections - as in
TMB protocols for horse radish peroxidase of the 1970's/80's.
"Defatting" is a lipid extraction step that when used on
well fixed tissue allows even penetration of stain and complete and
subtle differentiation in frozen sections of brain. The thicker
the section the more important this step becomes, as the unextracted
phospholipids are a barrier to penetration by aqueous solutions.
</div>
<div><x-tab> </x-tab></div>
<div>Well-dried frozen sections mounted onto slides can be defatted
by initial treatment in xylene or xylene substitute (10-30min
[minimum], with 2 changes of solution) followed by stepwise hydration
in alcohols [100%, 95%, 70%, 50%; 3- 5min each] down to water for
3-5min before staining. (You don't mention the concentration or
pH of your stain, both of which are important) After staining
and brief water rinse, virtually all differentiation is by
alcohol. Differentiation is most rapid in 70% and endpoint can
be checked by microscope (or by experienced eye) in 100% or the first
clearing agent. Two minutes in clean solutions of each alcohol
is a good starting point. Adjust number of changes of each
alcohol to optimize differentiation [e.g., 2x70%, 2x95, 2-3x100%]
depending on thickness of sections. Agitate gently for 1-20 sec
at the beginning of each new rinse, to assure even differentiation -
else the sections may be streaked, or lighter on top edge than on
bottom. Use a generous volume of solution for these thick,
large-area sections and place sections in alternate spaces in the
staining rack to get better flow of solution across the section
surface. Note: sections can be completely
de-stained by 1-24h in 70% alcohol, and then re-stained as
desired. So, 'too dark' sections need never be a problem.</div>
<div><br></div>
<div>A good alternative to xlyene-defatting is to place slides
directly into 70% alcohol and leave --<u>overnight</u>-- before
hydrating to water and staining as usual. Use10%formalin made
in 70%alcohol rather than buffer, if you think there is a problem of
underfixation. This helpful hint (supposedly from a Johns
Hopkins research lab, circa 1960) is useful when staining seems
uneven over large areas of thick frozen sections. </div>
<div><x-tab> </x-tab></div>
<div>For a complete discussion of staining brain sections for Nissl
substance, see: Simmons and Swanson, "The Nissl
Stain" in Neuroscience Protocols, 93-050-12-01-07, Elsevier
press, 1993. Reply to me by email and I can fax a copy if
you need one.</div>
<div><br></div>
<div>-Donna Simmons</div>
<div><br></div>
<div>>----------------------------------------<span
></span>------------------------------</div>
<div>>Date: 25 Sep 2000 17:43:22 -0500</div>
<div>>From: Lorraine Masse <lmasse@biosupport.com></div>
<div>>Subject: FW: Cresyl Violet Staining</div>
<div><br></div>
<div>>> I am using Cresyl Violet Acetate to stain serial
sections of macaque</div>
<div>>> brain. This tissue has been fixed in formalin for
over 6 months, frozen<br>
>> sectioned at 40 microns, and placed in PBS with 0.05% Sodium
Azide before<br>
>> mounting on slides. I have been running into problems
with fat<br>
>> displacement when I mount the slides onto coverslips.
The fat tends to<br>
>> spread all over the slide and causes problems when viewing
the slides<br>
>> under the microscope. The protocol for staining calls
for 2 minutes of<br>
>> hydration in deionized water, 1.5 minutes in the Cresyl
Violet Stain, 4</div>
<div>>> rinses in tap water for 30 seconds each, run through
alcohol at 80, 95,<br>
>> 100, and 100 percent for 30 seconds each, 2 minutes in
xylene twice, and<br>
>> coverslip. I have tried hydrating the tissue in
alcohol at 100, 95, 70,<br>
>> 50, and 35 percent before starting the deionized water
step, which caused<br>
>> the stain to be too dark for counting neurons. I have
also tried fixing<br>
>> the tissue in 40% formalin before starting the deionized
water step, which<br>
>> discolors the stain. Do you have any other ideas that
might be<br>
>> successful?<br>
>><br>
>> Thank you,</div>
<div>>> Sarah McGrath</div>
</body>
</html>