Re: [Histonet] cresyl violet staining on unfixed frozen brain sections

From:John Kiernan

15-30 seconds is rather fast for a critical
differentiation, unless you are handling individual
slides. Consider omitting the acetic acid or reducing 
its concentration, especially if you are staining 
several slides in a Coplin jar or rack.

When the neurons look right, or when they are slightly
overstained, quickly move the slides into the first of
three changes of 100% alcohol, agitate vigorously and
then go into the second 100%. Alcohol-water mixtures
extract most dyes more rapidly than either water or 
100% alcohol used alone. 

For what it's worth, I prefer toluidine blue or neutral
red to cresyl violet for Nissl staining, partly because
the dye solutions can be kept and re-used for years.
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
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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