[Histonet] cresyl violet staining on unfixed frozen brain sections

[Jo Dee Fish]

Hi Hector,
I differentiate in 95% Ethanol (can add 0.1% acetic acid, but not 
necessary), nothing less.  It seems that going into a lower 
concentration of ethanol destains the slides.  After 95%, I continue 
dehydration in 100% (3x) 10 dips each, then 2 minutes in xylene, 20 
dips in fresh xylene, and coverslip out of another fresh xylene. 
Works great!
Jo Dee

At 5:40 PM +0100 2/11/05, Hector Gonzalez Pardo wrote:
>Hi everyone!
>I am trying to perform Nissl staining using cresyl violet acetate on 
>30 micrometer-tick cryostat sections from rat brain (I usually don't 
>need gelatinized slides). After re-hydriting the sections in 
>alcohols (70%, 80%, 96% and 100%), I immerse the sections for 10-15 
>min in a solution of cresyl violet acetate (Sigma, 0,5 % at pH 4). I 
>differentiate the sections using 70% ethanol with a few drops of 
>glacial acetic acid (for abut 15-30 secs.), but when I try to 
>deydrate the sections using 80%, 96% and 100 % ethanol, I always 
>lose the staing. Finally, I get a blueish pale-looking Nissl 
>staining on my sections, and I cannot see anything. Does anyone know 
>how to solve it? I've tried to first de-fat the sections using 
>ethanol for a few minutes but I did'nt work. Thank you!
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Jo Dee Fish
Research Technologist III
Gladstone Institute of Cardiovascular Disease

Telephone: (415) 734-5766
Fax: (415) 355-0230
E-mail: jfish <@t> gladstone.ucsf.edu

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