[Histonet] cresyl violet staining on unfixed frozen brain sections

[Hector Gonzalez Pardo]

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!