FW: Cresyl Violet Staining

From:Donna Simmons <dsimmons@usc.edu>

<!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>
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