[Histonet] Problem with cresyl violet staining on unfixed frozen brain sections

From:Gayle Callis


You may be taking out too much stain with your alcohol rinsing steps or the 
acetic acid step (this may be too long and acid too concentrated).   Fat is 
probably not your problem but too much stain removal.  See B-2 steps for 
that comment.  Your sections are thick, so staining them may take a bit 
more patience.  You say unfixed frozen sections, so you are letting the 
absolute alcohol fix the fresh, unfixed brain frozen section, then rehydrate?

These are some staining hints we have with our method:

Variations to Cresyl violet

A.        If staining is too dark, staining time can be increased.  If too 
dark, acetic acid water can be used to differentiate out stain       (1 - 
2  ml acetic acid in 250 ml distilled water) with 1 to 2 very quick dips, 
rinse with water, dehydrate, clear and coverslip.        Use acid water 
step carefully, you can overdo removal of stain.

B.         To lighten staining
             1)  Staining time can be decreased to 45 sec, rinsed with 70% 
ethanol 3 dips then into 95% ethanol.
             2)  Water rinses, acetic acid water dips, and alcohols will 
remove stain

Good luck

At 09:40 AM 2/11/2005, you 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!

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

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